Bipyridine and bipyrimidine gold(iii) dithiocarbamate complexes and methods of treating cancer

ABSTRACT

A gold(III) complex of formula (I) or formula (II) 
     
       
         
         
             
             
         
       
         
         
           
             wherein R 1  and R 2  are each independently a hydrogen, an optionally substituted alkyl, an optionally substituted cycloalkyl, an optionally substituted arylalkyl, or an optionally substituted aryl; R 3  and R 4  are each independently a hydrogen, an optionally substituted alkyl, an optionally substituted cycloalkyl, an optionally substituted arylalkyl, an optionally substituted aryl, an optionally substituted heterocyclyl, an optionally substituted alkoxy, a hydroxyl, a halo, a nitro, a cyano, a N-monosubstituted amino group, or a N,N-disubstituted amino group; and X is Cl, Br, or I. A pharmaceutical composition containing the gold(III) complex of formula (I) or (II), and a method of treating cancer are included.

STATEMENT OF ACKNOWLEDGEMENT

This research was supported in part by grant IN171005 from the King FahdUniversity of Petroleum and Minerals.

STATEMENT REGARDING PRIOR DISCLOSURE BY THE INVENTORS

Aspects of this technology are described in an article “Potent In Vitroand In Vivo Anticancer Activity of New Bipyridine and Bipyrimidine Gold(III) Dithiocarbamate Derivatives” published in Cancers, 2019, 11, 474,on Apr. 4, 2019, which is incorporated herein by reference in itsentirety.

BACKGROUND OF THE INVENTION Technical Field

The present disclosure relates to bipyridine and bipyrimidine gold(III)dithiocarbamate-containing complexes with anticancer or antitumorproperties, and pharmaceutical compositions and uses thereof.

Description of the Related Art

The “background” description provided herein is for the purpose ofgenerally presenting the context of the disclosure. Work of thepresently named inventors, to the extent it is described in thisbackground section, as well as aspects of the description which may nototherwise qualify as prior art at the time of filing, are neitherexpressly or impliedly admitted as prior art against the presentinvention.

Cisplatin and a few related platinum compounds, such as carboplatin andoxaliplatin, are common anticancer agents, but their use often causessignificant toxicity and leads to drug resistance. See Spreckelmeyer S.;Orvig C.; Casini A. Cellular transport mechanisms of cytotoxicmetallodrugs: an overview beyond cisplatin. Molecules 2014.19(10),15584-15610; Cappetta D.; Rossi F.; Piegari E.; Quaini F.;Berrino L.; Urbanek K.; De A. A. Doxorubicin targets multiple players: Anew view of an old problem. Pharmacol Res 2018. 127,4-14; Galluzzi L.;Vitale I.; Michels J.; Brenner C.; Szabadkai G.; Harel-Bellan A.;Castedo M.; Kroemer G. Systems biology of cisplatin resistance: past,present and future. Cell Death Dis 2014. 5,e1257; and Dilruba S.;Kalayda G. V. Platinum-based drugs: past, present and future. CancerChemother Pharmacol 2016. 77,1103-1124, each incorporated herein byreference in their entirety. Therefore, other metallodrugs containingplatinum or non-platinum metals, such as ruthenium, palladium, titanium,gold and copper, have been investigated. See Nardon C.; Fregona D.Editorial: Throwing Light on Recent Advances on Metallodrugs: FromDeemed Poisons to a Striking Hope for the Future. Curr Med Chem 2018.25(4),434-436; Lazarevic T.; Rilak A.; Bugarcic Z. D. Platinum,palladium, gold and ruthenium complexes as anticancer agents: Currentclinical uses, cytotoxicity studies and future perspectives. Eur J MedChem 2017. 142,8-31; Soldevila-Barreda J. J.; Sadler P. J. Approaches tothe design of catalytic metallodrugs. Curr Opin Chem Biol 2015.25,172-183; and Casini A.; Sun R. W.; Ott I. Medicinal Chemistry of GoldAnticancer Metallodrugs. Met Ions Life Sci 2018. 18.pii,books/9783110470734-013, each incorporated herein by reference intheir entirety. In particular, gold(I) and gold(III) complexes have beenfound to have anticancer effects in vitro and in vivo. See, Nardon C.;Fregona D. Editorial: Throwing Light on Recent Advances on Metallodrugs:From Deemed Poisons to a Striking Hope for the Future. Curr Med Chem2018. 25(4),434-436; Casini A. Cellular transport mechanisms ofcytotoxic metallodrugs: an overview beyond cisplatin. Molecules 2014.19(10),15584-15610; and Bertrand B.; Williams M. R. M.; Bochmann M.Gold(III) Complexes for Antitumor Applications: An Overview. Chemistry2018. 24(46),11840-11851, each incorporated herein by reference in theirentirety.

Gold(I) and gold(III) complexes have a variety of mechanisms of action,including inhibition of the enzyme thioredoxin reductase (TrxR),increased generation of reactive oxygen species (ROS), proteasomeinhibition, interaction with DNA, alteration of the cell cycle phasesand modulation of kinases. See Bertrand B.; Williams M. R. M.; BochmannM. Gold(III) Complexes for Antitumor Applications: An Overview.Chemistry 2018. 24(46),11840-11851; Celegato M.; Borghese C.; CasagrandeN.; Mongiat M.; Kahle X. U.; Paulitti A.; Spina M.; Colombatti A.;Aldinucci D. Preclinical activity of the repurposed drug Auranofin inclassical Hodgkin lymphoma. Blood 2015. 126,1394-1397; Aldinucci D.;Lorenzon D.; Stefani L.; Giovagnini L.; Colombatti A.; Fregona D.Antiproliferative and apoptotic effects of two new gold(III)methylsarcosinedithiocarbamate derivatives on human acute myeloidleukemia cells in vitro. Anticancer Drugs 2007. 18,323-332; Milacic V.;Chen D.; Ronconi L.; Landis-Piwowar K. R.; Fregona D.; Dou Q. P. A novelanticancer gold(III) dithiocarbamate compound inhibits the activity of apurified 20S proteasome and 26S proteasome in human breast cancer cellcultures and xenografts. Cancer Res 2006. 66,10478-10486; Gratteri P.;Massai L.; Michelucci E.; Rigo R.; Messori L.; Cinellu M. A.; MusettiC.; Sissi C.; Bazzicalupi C. Interactions of selected gold(III)complexes with DNA G quadruplexes. Dalton Trans 2015. 44(8),3633-3639;Coronnello M.; Marcon G.; Carotti S.; Caciagli B.; Mini E.; Mazzei T.;Orioli P.; Messori L. Cytotoxicity, DNA damage, and cell cycleperturbations induced by two representative gold(III) complexes in humanleukemic cells with different cisplatin sensitivity. Oncol Res 2000.12(9-10),361-370; and Saggioro D.; Rigobello M. P.; Paloschi L.; FoldaA.; Moggach S. A.; Parsons S.; Ronconi L.; Fregona D.; Bindoli A.Gold(III)-dithiocarbamato complexes induce cancer cell death triggeredby thioredoxin redox system inhibition and activation of ERK pathway.Chem Biol 2007. 14,1128-1139, each incorporated herein by reference intheir entirety. These multifaceted modes of action enable gold complexesto exert potent cytotoxicity against cancer cells, includingmultidrug-resistant tumor cells. See Nardon C.; Fregona D. Editorial:Throwing Light on Recent Advances on Metallodrugs: From Deemed Poisonsto a Striking Hope for the Future. Curr Med Chem 2018. 25(4),434-436;and Bertrand B.; Williams M. R. M.; Bochmann M. Gold(III) Complexes forAntitumor Applications: An Overview. Chemistry 2018. 24(46),11840-11851,each incorporated herein by reference in its entirety. However, gold andother heavy metals such as platinum also react with thesulfur-containing amino acids cysteine (a thiol) and methionine (athioether), generating metal-protein adducts that can be nephrotoxic.See Bertrand B.; Williams M. R. M.; Bochmann M. Gold(III) Complexes forAntitumor Applications: An Overview. Chemistry 2018. 24(46),11840-11851.

The use of dithiocarbamate as a chelating ligand can potentially preventinteractions between the metal center of anticancer drugs andthiol-containing biomolecules. Through its sulfur atoms, dithiocarbamatecoordinates metal ions, thereby stabilizing metal drugs and reducinginteractions with biomolecules. See Nardon C.; Fregona D. Editorial:Throwing Light on Recent Advances on Metallodrugs: From Deemed Poisonsto a Striking Hope for the Future. Curr Med Chem 2018. 25(4),434-436;and Marzano C.; Ronconi L.; Chiara F.; Giron M. C.; Faustinelli I.;Cristofori P.; Trevisan A.; Fregona D. Gold(III)-dithiocarbamatoanticancer agents: activity, toxicology and histopathological studies inrodents. Int J Cancer 2011. 129(2),487-496, each incorporated herein byreference in their entirety. Gold(III) dithiocarbamate complexes havebeen reported to have potent in vitro anticancer activity against acutemyeloid leukemia cells and prostate cancer cells and low toxicity intumor-bearing mice. See Aldinucci D.; Lorenzon D.; Stefani L.;Giovagnini L.; Colombatti A.; Fregona D. Antiproliferative and apoptoticeffects of two new gold(III) methylsarcosinedithiocarbamate derivativeson human acute myeloid leukemia cells in vitro. Anticancer Drugs 2007.18,323-332; and Cattaruzza L.; Fregona D.; Mongiat M.; Ronconi L.;Fassina A.; Colombatti A.; Aldinucci D. Antitumor activity ofgold(III)-dithiocarbamato derivatives on prostate cancer cells andxenografts. Int J Cancer 2011. 128,206-215, each incorporated herein byreference in their entirety. Second generation gold(III) dithiocarbamatecomplexes which are derivatives of oligopeptides (peptidomimetics) havealso been made to improve the delivery and cellular uptake of thesecompounds. See Kouodom M. N.; Ronconi L.; Celegato M.; Nardon C.;Marchio L.; Dou Q. P.; Aldinucci D.; Formaggio F.; Fregona D. Toward theselective delivery of chemotherapeutics into tumor cells by targetingpeptide transporters: tailored gold-based anticancer peptidomimetics. JMed Chem 2012. 55(5), 2212-2226, incorporated herein by reference in itsentirety. These peptidomimetics were designed to target the peptidetransporters PEPT1 and PEPT2 that are unregulated in several tumortypes, and showed promising anticancer activity in different tumormodels, including breast and prostate cancer. See Kouodom M. N.; RonconiL.; Celegato M.; Nardon C.; Marchio L.; Dou Q. P.; Aldinucci D.;Formaggio F.; Fregona D. Toward the selective delivery ofchemotherapeutics into tumor cells by targeting peptide transporters:tailored gold-based anticancer peptidomimetics. J Med Chem 2012.55(5),2212-2226; Nardon C.; Schmitt S. M.; Yang H.; Zuo J.; Fregona D.;Dou Q. P. Gold(III)-dithiocarbamato peptidomimetics in the forefront ofthe targeted anticancer therapy: preclinical studies against humanbreast neoplasia. PLoS One 2014. 9(1),e84248; and Celegato M.; FregonaD.; Mongiat M.; Ronconi L.; Borghese C.; Canzonieri V.; Casagrande N.;Nardon C.; Colombatti A.; Aldinucci D. Preclinical activity ofmultiple-target gold(III)-dithiocarbamato peptidomimetics in prostatecancer cells and xenografts. Future Med Chem 2014. 6(11),1249-1263, eachincorporated herein by reference in their entirety.

In parallel research, other metallodrugs with anticancer activity,including gold(I) and gold(III) complexes have been designed andsynthesized. See Altaf M.; Monim-ul-Mehboob M.; Seliman A. A.; SohailM.; Wazeer M. I.; Isab A. A.; Li L.; Dhuna V.; Bhatia G.; Dhuna K.Synthesis, characterization and anticancer activity of gold(I) complexesthat contain tri-tert-butylphosphine and dialkyl dithiocarbamateligands. Eur J Med Chem 2015. 95,464-472; Altaf M.; Monom-ul-Mehboob M.;Selimam A. A.; Isab A. A.; Dhuna V.; Bhatia G.; Dhuna K. Synthesis,X-ray Structures, Spectroscopic Analysis and Anticancer Activity ofNovel Gold(I) Carbene Complexes. Journal of Organometallic Chemistry2014. 765,68-79; and Al-Jaroudi S. S.; Altaf M.; Al-Saadi A. A.; KawdeA. N.; Altuwaijri S.; Ahmad S.; Isab A. A. Synthesis, characterizationand theoretical calculations of(1,2-diaminocyclohexane)(1,3-diaminopropane)gold(III) chloridecomplexes: in vitro cytotoxic evaluations against human cancer celllines. Biometals 2015. 28(5),827-844, each incorporated herein byreference in their entirety. Together, these two research groupsproduced new bipyridine gold(III) dithiocarbamate complexes withnitrogen and sulfur donor ligands which are cytotoxic incisplatin-resistant ovarian carcinoma cells as well as in p53-defectivecancer cells of different tumor types, and wherein certain complexes areless cytotoxic in non-cancer human mesenchymal stromal cells than incancer cells. See Altaf M.; Monim-ul-Mehboob M.; Kawde A. N.; Corona G.;Larcher R.; Ogasawara M.; Casagrande N.; Celegato M.; Borghese C.;Siddik Z. H.; Aldinucci D.; Isab A. A. New bipyridine gold(III)dithiocarbamate-containing complexes exerted a potent anticanceractivity against cisplatin-resistant cancer cells independent of p53status. Oncotarget 2017. 8(1),490-505, incorporated herein by referencein its entirety. However, new gold complexes are needed with improvedpotency, antitumor properties, and with lower toxicity.

In view of the forgoing, one objective of the present disclosure is toprovide safe and potent therapeutic complexes with low- andsub-micromolar antiproliferative activity based on bipyridine orbipyrimidine gold(III) dithiocarbamate-containing complexes, apharmaceutical composition containing the gold(III) complexes, and amethod for treating cancer with the gold(III) complexes.

BRIEF SUMMARY OF THE INVENTION

Accordingly, it is one object of the present invention to providebipyridine and bipyrimidine gold(III) dithiocarbamate-containingcomplexes, which are non-toxic, have excellent pharmacologic propertieswith low- and sub-micromolar antiproliferative activity, and whichremain effective even in multiple drug resistant cancers.

It is another object of the present invention to provide pharmaceuticalcompositions containing the gold(III) complexes.

It is yet another object of the present invention to provide methods fortreating cancer with the gold(III) complexes.

Thus, the present invention provides:

A gold(III) complex of formula (I),

or a pharmaceutically acceptable salt, solvate, tautomer, orstereoisomer thereof,

wherein:

R¹ and R² are each independently a hydrogen, an optionally substitutedalkyl, an optionally substituted cycloalkyl, an optionally substitutedarylalkyl, or an optionally substituted aryl;

R³ and R⁴ are each independently a hydrogen, an optionally substitutedalkyl, an optionally substituted cycloalkyl, an optionally substitutedarylalkyl, an optionally substituted aryl, an optionally substitutedheterocyclyl, an optionally substituted alkoxy, a hydroxyl, a halo, anitro, a cyano, a N-monosubstituted amino group, or a N,N-disubstitutedamino group; and

X is Cl, Br, or I.

In some embodiments, R¹ and R² are each independently a C₁ to C₈ alkylor a C₇ to C₁₂ arylalkyl.

In some embodiments, R¹ and R² are each methyl, ethyl, or benzyl.

In some embodiments, R³ and R⁴ are each hydrogen.

In some embodiments, X is Cl.

In some embodiments, the gold(III) complex is selected from the groupconsisting of

A pharmaceutical composition, which includes the gold(III) complex offormula (I) and a pharmaceutically acceptable carrier and/or excipient.

In some embodiments, the gold(III) complex of formula (I) is present inthe pharmaceutical composition in a concentration of 1 to 50 μM,relative to a total volume of the pharmaceutical composition.

A method for treating cancer in a subject that includes administering tothe subject a therapeutically effective amount of the gold(III) complexof formula (I), wherein the cancer is at least one selected from thegroup consisting of bone cancer, lung cancer, prostate cancer, breastcancer, ovarian cancer, and cervical cancer.

In some embodiments, the therapeutically effective amount of thegold(III) complex of formula (I) is from 0.01 to 25 mg/kg of thegold(III) complex of formula (I) per body weight of the subject.

A gold(III) complex of formula (II),

or a pharmaceutically acceptable salt, solvate, tautomer, orstereoisomer thereof,

wherein:

R¹ and R² are each independently a hydrogen, an optionally substitutedalkyl, an optionally substituted cycloalkyl, an optionally substitutedarylalkyl, or an optionally substituted aryl;

R³ and R⁴ are each independently an optionally substituted alkyl, anoptionally substituted cycloalkyl, an optionally substituted arylalkyl,an optionally substituted aryl, an optionally substituted heterocyclyl,an optionally substituted alkoxy, a hydroxyl, a halo, a nitro, a cyano,a N-monosubstituted amino group, or a N,N-disubstituted amino group; and

X is Cl, Br, or I.

In some embodiments, R¹ and R² are each independently a C₁ to C₈ alkylor a C₇ to C₁₂ arylalkyl.

In some embodiments, R¹ and R² are each methyl, ethyl, or benzyl.

In some embodiments, R³ and R⁴ are each hydroxyl.

In some embodiments, X is Cl.

In some embodiments, the gold(III) complex of formula (II) is selectedfrom the group consisting of

A pharmaceutical composition, that includes the gold(III) complex offormula (II) and a pharmaceutically acceptable carrier and/or excipient.

In some embodiments, the gold(III) complex of formula (II) is present inthe pharmaceutical composition in a concentration of 1 to 50 μM,relative to a total volume of the pharmaceutical composition.

A method for treating cancer in a subject, that includes administeringto the subject a therapeutically effective amount of the gold(III)complex of formula (II), wherein the cancer is at least one selectedfrom the group consisting of bone cancer, lung cancer, prostate cancer,breast cancer, ovarian cancer, and cervical cancer.

In some embodiments, the therapeutically effective amount of thegold(III) complex of formula (II) is from 0.01 to 25 mg/kg of thegold(III) complex of formula (II) per body weight of the subject.

The foregoing paragraphs have been provided by way of generalintroduction, and are not intended to limit the scope of the followingclaims. The described embodiments, together with further advantages,will be best understood by reference to the following detaileddescription taken in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete appreciation of the disclosure and many of the attendantadvantages thereof will be readily obtained as the same becomes betterunderstood by reference to the following detailed description whenconsidered in connection with the accompanying drawings, wherein:

FIGS. 1A-1H illustrate chemical structures of gold(III) complexes C1-C8.

FIG. 2A is a graph showing the percentage of annexin-V-positive cellsand Annexin-V-propidium iodide (PI)-positive cells using PC3 cellscultured in complete medium alone or with complex C6, at thehalf-maximal inhibitory concentration (IC50), at IC25, or IC75, for 24h, illustrating the induced apoptosis/cell cycle block from complex C6;

FIG. 2B shows representative flow cytometry plots of the Annexin-V-FITCand propidium iodide (PI) assay of apoptosis evaluated by flow cytometryusing the conditions of FIG. 1A, where the percentages of stained cellsare reported;

FIG. 2C is a graph illustrating Caspase 3,7 activation assay evaluatedby flow cytometry with fluorochrome-labeled inhibitors of caspases(FLICA) in terms of mean fluorescence intensity (MFI) of FLICA under theconditions of FIG. 1A;

FIG. 2D shows representative flow cytometry histograms of caspase 3,7activation by C6 treatment according to FIG. 1C where the percentage ofFLICA-positive cells is reported;

FIG. 2E shows the cell cycle progression determined by PI staining interms of percentage distribution of PC3 cells in different cell cyclephases under the conditions of FIG. 1A;

FIG. 2F shows representative flow cytometry histograms of cell cycleprogression of FIG. 3E.

FIG. 3A is a graph illustrating reactive oxygen species (ROS)generation-by PC3 cells in terms of mean fluorescence intensity (MFI)when exposed to medium alone or complex C6 at its IC50 and IC75 for 24h, in the presence or absence of the ROS scavenger N-acetyl cysteine(NAC) (added 30 minutes before drug treatment), where ROS were detectedusing H2DCFDA, showing that the complex C6 increases ROS generation;

FIG. 3B shows representative flow cytometry histograms of generated ROSfrom FIG. 1A;

FIG. 3C is a graph showing the number of viable cells 24 h aftertreatment with C6 and NAC (NAC, added 30 minutes before drug treatment),evaluated by trypan blue dye exclusion;

FIG. 3D is a graph showing histone H2A.X phosphorylation (YH2A.X) as ameasure of double-stranded DNA breaks, detected with FITC anti-H2A.XPhospho (Ser139) antibody in terms of YH2A.X (MFI) levels after a 24 htreatment of PC3 cells with C6;

FIG. 3E is a representative flow cytometry histogram of YH2A.X of FIG.3D;

FIG. 3F is a graph illustrating thioredoxin reductase (TrxR) activityafter a 12 h treatment with C6, normalized to control (medium);

FIG. 3G is a graph illustrating proteasome activity after 12 h treatmentwith C6, evaluated with the 20S-Proteasome Assay kit, normalized tocontrol (medium).

FIG. 4A is a graph illustrating results from a scratch assay whereconfluent monolayers of PC3 cells were treated or not with complex C6(IC50) for 3 h in complete medium, “wounded” by scraping, then culturedin low serum medium and photographed every 12 h for up to 36 h, with thedata expressed in terms of cell-free area remaining over time as cellsmigrated into the wound, normalized to time 0 (the mean and SD of threeexperiments each done in triplicate; *P<0.05, Student's t test), showingthat the complex C6 reduces cell migration;

FIG. 4B shows representative phase contrast photomicrographs, originalmagnification 4×, from the scratch assay of FIG. 4A;

FIG. 4C is a graph showing growth of xenografts in nude mice inoculatedwith PC3 cells (3×106 cells/animal) and treated intratumorally with C6(2.5 mg/kg) (n=5) or vehicle (n=5), values are expressed as mean and SD,Student's t test, *P<0.0001;

FIG. 4D is a graph of the body weights of xenografted mice (n=5 pergroup) from FIG. 4C.

FIGS. 5A-5D are voltammograms for the interaction of complex Cl withlysozyme in 0.1 M phosphate buffer (pH 6.8) or in control experiments(C1 in double-distilled water);

FIGS. 6A-6D are voltammograms for the interaction of C2 with lysozyme in0.1 M phosphate buffer (pH 6.8) or in control experiments (C2 indouble-distilled water);

FIGS. 7A-7D are voltammograms for the interaction of C3 with lysozyme in0.1 M phosphate buffer (pH 6.8) or in control experiments (C3 indouble-distilled water);

FIGS. 8A-8D are voltammograms for the interaction of complex C4 withlysozyme in 0.1 M phosphate buffer (pH 6.8) or in control experiments(C4 in double-distilled water);

FIGS. 9A-9D are voltammograms for the interaction of complex C5 with 0.5mM tryptophan in 0.1 M phosphate buffer (pH 6.8) or control experiments(0.5 mM tryptophan alone or C5 alone with varying volumes of ethanol);

FIGS. 10A-10D are voltammograms for the interaction of complex C6 with0.5 mM tryptophan in 0.1 M phosphate buffer (pH 6.8) or controlexperiments (0.5 mM tryptophan alone or C6 alone with varying volumes ofethanol);

FIGS. 11A-11D are voltammograms for the interaction of complex C7 with0.5 mM tryptophan in 0.1 M phosphate buffer (pH 6.8) or controlexperiments (0.5 mM tryptophan alone or C7 alone with varying volumes ofethanol);

FIGS. 12A-12D are voltammograms for the interaction of complex C8 with0.5 mM tryptophan in 0.1 M phosphate buffer (pH 6.8) or controlexperiments (0.5 mM tryptophan alone or C8 alone with varying volumes ofethanol);

FIGS. 13A-13F are voltammograms for the interaction of complex C1 with0.5 mM guanine in 0.1 M phosphate buffer (pH 6.8) or control experiments(0.5 mM guanine alone with varying volumes of ethanol or C1 alone);

FIGS. 14A-14F are voltammograms for the interaction of complex C5 with0.5 mM guanine in 0.1 M phosphate buffer (pH 6.8) or control experiments(0.5 mM guanine alone with varying volumes of ethanol or C5 alone);

FIG. 15 is a graph illustrating the uptake of selected gold(III)complexes by PC3 cells, where PC3 cells (1×10⁶ cells/dish) were treatedfor 2 h with 3 μM C4, C5, C6 or C7, and internalized gold was determinedby ICP mass spectrometry, with results presented as means and SD ofthree independent experiments;

FIG. 16 is a graph illustrating the growth inhibition curves for C6 inPC3 prostate cancer cells and adipose-derived stromal cells (ADSCs),where cell viability was determined with the MTT assay after 72 h drugtreatment, and the results are presented as means and SD for threereplicate wells from three independent experiments.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Embodiments of the present disclosure will now be described more fullyhereinafter with reference to the accompanying drawings, in which some,but not all embodiments of the disclosure are shown.

Definitions

As used herein, the terms “compound”, “complex”, and “product” are usedinterchangeably, and are intended to refer to a chemical entity, whetherin the solid, liquid or gaseous phase, and whether in a crude mixture orpurified and isolated. In the present disclosure, the phrase “gold (III)complex” or “gold (III) complexes” may refer to the gold(III) complex offormula (I), the gold(III) complex of formula II, or both, unlessotherwise specified.

As used herein, the term “mononuclear” refers to coordination complexescontaining a single metal atom (ion) in a single coordination sphere.While the term “binuclear” refers to coordination compounds containingtwo metal atoms (ions) in a single coordination sphere. The two atomsmay be held together through direct metal-metal bonds, through bridgingligands, or both.

Throughout the specification and the appended claims, a given chemicalformula or name shall encompass all stereo and optical isomers andracemates thereof where such isomers exist. Unless otherwise indicated,all chiral (enantiomeric and diastereomeric) and racemic forms arewithin the scope of the disclosure. Many geometric isomers of C═C doublebonds, C═N double bonds, ring systems, and the like can also be presentin the complexes, and all such stable isomers are contemplated in thepresent disclosure. Cis- and trans- (or E- and Z-) geometric isomers ofthe complexes of the present disclosure are described and may beisolated as a mixture of isomers or as separated isomeric forms. Thepresent complexes can be isolated in optically active or racemic forms.Optically active forms may be prepared by resolution of racemic forms orby synthesis from optically active starting materials. All processesused to prepare complexes of the present disclosure and intermediatesmade therein are considered to be part of the present disclosure. Whenenantiomeric or diastereomeric products are prepared, they may beseparated by conventional methods, for example, by chromatography,fractional crystallization, or through the use of a chiral agent.Depending on the process conditions the end products of the presentdisclosure are obtained either in free (neutral) or salt form. Both thefree form and the salts of these end products are within the scope ofthe disclosure. If so desired, one form of a compound may be convertedinto another form. A free base or acid may be converted into a salt; asalt may be converted into the free compound or another salt; a mixtureof isomeric complexes of the present disclosure may be separated intothe individual isomers. Complexes of the present disclosure, free formand salts thereof, may exist in multiple tautomeric forms, in whichhydrogen atoms are transposed to other parts of the molecules and thechemical bonds between the atoms of the molecules are consequentlyrearranged. It should be understood that all tautomeric forms, insofaras they may exist, are included within the disclosure. Further, a givenchemical formula or name shall encompass all conformers, rotamers, orconformational isomers thereof where such isomers exist. Differentconformations can have different energies, can usually interconvert, andare very rarely isolatable. There are some molecules that can beisolated in several conformations. For example, atropisomers are isomersresulting from hindered rotation about single bonds where the stericstrain barrier to rotation is high enough to allow for the isolation ofthe conformers. It should be understood that all conformers, rotamers,or conformational isomer forms, insofar as they may exist, are includedwithin the present disclosure.

As used herein, the term “solvate” refers to a physical association of acompound of this disclosure with one or more solvent molecules, whetherorganic or inorganic. This physical association includes hydrogenbonding. In certain instances, the solvate will be capable of isolation,for example when one or more solvent molecules are incorporated in thecrystal lattice of the crystalline solid. The solvent molecules in thesolvate may be present in a regular arrangement and/or a non-orderedarrangement. The solvate may comprise either a stoichiometric ornonstoichiometric amount of the solvent molecules. Solvate encompassesboth solution phase and isolable solvates. Exemplary solvents include,but are not limited to, water, methanol, ethanol, n-propanol,isopropanol, n-butanol, isobutanol, tert-butanol, ethyl acetate andother lower alkanols, glycerine, acetone, dichloromethane (DCM),dimethyl sulfoxide (DMSO), dimethyl acetate (DMA), dimethylformamide(DMF), isopropyl ether, acetonitrile, toluene, N-methylpyrrolidone(NMP), tetrahydrofuran (THF), tetrahydropyran, other cyclic mono-, di-and tri-ethers, polyalkylene glycols (e.g., polyethylene glycol,polypropylene glycol, propylene glycol), and mixtures thereof insuitable proportions. Exemplary solvates include, but are not limitedto, hydrates, ethanolates, methanolates, isopropanolates and mixturesthereof. Methods of solvation are generally known to those of ordinaryskill in the art.

The present disclosure is intended to include all isotopes of atomsoccurring in the present complexes. Isotopes include those atoms havingthe same atomic number but different mass numbers. By way of generalexample and without limitation, isotopes of hydrogen include deuteriumand tritium. Isotopes of carbon include ¹³C and ¹⁴C.Isotopically-labeled compounds of the disclosure can generally beprepared by conventional techniques known to those skilled in the art orby processes analogous to those described herein, using an appropriateisotopically-labeled reagent in place of the non-labeled reagentotherwise employed.

As used herein, “pharmaceutically acceptable salt” refers to derivativesof the disclosed complexes wherein the parent complex is modified bymaking acid or base salts thereof. Examples of pharmaceuticallyacceptable salts include, but are not limited to, mineral or organicacid salts of basic groups such as amines; and alkali or organic saltsof acidic groups such as carboxylic acids. The pharmaceuticallyacceptable salts include the conventional non-toxic salts or thequaternary ammonium salts of the parent compound formed, for example,from non-toxic inorganic or organic acids. For example, suchconventional non-toxic salts include those derived from inorganic acidssuch as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, andnitric; and the salts prepared from organic acids such as acetic,propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric,ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic,benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric,toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, andisethionic, and the like. The pharmaceutically acceptable salts of thepresent disclosure can be synthesized from the parent complex thatcontains a basic or acidic moiety by conventional chemical methods.Generally, such salts can be prepared by reacting the free acid or baseforms of these compounds with a stoichiometric amount of the appropriatebase or acid in water or in an organic solvent, or in a mixture of thetwo; generally, non-aqueous media like ether, ethyl acetate, ethanol,isopropanol, or acetonitrile are preferred. Lists of suitable salts arefound in Remington's Pharmaceutical Sciences, 18th Edition, MackPublishing Company, Easton, Pa. (1990), the disclosure of which ishereby incorporated by reference.

As used herein, the term “alkyl” unless otherwise specified refers toboth branched and straight chain aliphatic (non-aromatic) hydrocarbonswhich may be primary, secondary, and/or tertiary hydrocarbons typicallyhaving 1 to 32 carbon atoms (e.g., C₁, C₂, C₃, C₄, C₅, C₆, C₇, C₈, C₉,C₁₀, C₁₁, C₁₂, C₁₃, C₁₄, etc.) and specifically includes, but is notlimited to, saturated alkyl groups such as methyl, ethyl, propyl,isopropyl, butyl, isobutyl, t-butyl, pentyl, isopentyl, neopentyl,hexyl, isohexyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl,2-ethylhexyl, heptyl, octyl, nonyl, 3,7-dimethyloctyl, decyl, undecyl,dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl,octadecyl, nonadecyl, eicosyl, guerbet-type alkyl groups (e.g.,2-methylpentyl, 2-ethylhexyl, 2-proylheptyl, 2-butyloctyl,2-pentylnonyl, 2-hexyldecyl, 2-heptylundecyl, 2-octyldodecyl,2-nonyltridecyl, 2-decyltetradecyl, and 2-undecylpentadecyl), as well asunsaturated alkenyl and alkynyl variants such as vinyl, allyl,1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl,2-pentenyl, 3-pentenyl, 4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl,4-hexenyl, 5-hexenyl, oleyl, linoleyl, and the like.

The term “cycloalkyl” refers to cyclized alkyl groups. Exemplarycycloalkyl groups include, but are not limited to, saturated cycloalkylgroups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,norbornyl, and adamantyl, branched cycloalkyl groups such as1-methylcyclopropyl and 2-methylcyclopropyl, and cycloalkenyl groupssuch as cyclobutenyl, cyclopentenyl, and cyclohexenyl.

The term “aryl” means a carbocyclic aromatic monocyclic group containing6 carbon atoms which may be further fused to a second 5- or 6-memberedcarbocyclic group which may be aromatic, saturated or unsaturated. Arylincludes, but is not limited to, phenyl, anthracenyl, indanyl,1-naphthyl, 2-naphthyl, and tetrahydronaphthyl. The fused aryls may beconnected to another group either at a suitable position on thecycloalkyl/cycloalkenyl ring or the aromatic ring.

The term “arylalkyl”, as used herein, refers to a straight or branchedchain alkyl moiety (as defined above) that is substituted by an arylgroup (as defined above), examples of which include, but are not limitedto, benzyl, phenethyl, 2-methylbenzyl, 3-methylbenzyl, 4-methylbenzyl,2,4-dimethylbenzyl, 2-(4-ethylphenyl)ethyl, 3-(3-propylphenyl)propyl,and the like.

The term “alkoxy” refers to an —O-alkyl group. Example alkoxy groupsinclude, but are not limited to, methoxy, ethoxy, propoxy (e.g.,n-propoxy and isopropoxy), and t-butoxy.

As used herein, the term “heterocycle” or “heterocyclyl” is intended tomean a stable 3-, 4-, 5-, 6-, or 7-membered monocyclic, 7-, 8-, 9-, 10-,or 11-membered bicyclic, or 7-, 8-, 9-, 10-, 11-, 12-, 13-, or14-membered polycyclic heterocyclic ring that is saturated, partiallyunsaturated, or fully unsaturated, and that contains carbon atoms and 1,2, 3 or 4 heteroatoms independently selected from the group consistingof N, O and S; and including any bicyclic or polycyclic group in whichany of the above-defined heterocyclic rings is fused to a carbocyclicring, the carbocyclic ring being either saturated, unsaturated, oraromatic (e.g., a benzene ring). The nitrogen and sulfur heteroatoms mayoptionally be oxidized (i.e., N→0 and S(O)_(p), wherein p is 0, 1 or 2).The nitrogen atom may be substituted or unsubstituted (i.e., N or NRwherein R is H or another substituent, if defined). The heterocyclicring may be attached to its pendant group at any heteroatom or carbonatom that results in a stable structure. The heterocyclic ringsdescribed herein may be substituted on carbon or on a nitrogen atom ifthe resulting compound is stable. A nitrogen in the heterocycle mayoptionally be quaternized. It is preferred that when the total number ofS and O atoms in the heterocycle exceeds 1, then these heteroatoms arenot adjacent to one another. It is preferred that the total number of Sand O atoms in the heterocycle is not more than 1. When the term“heterocycle” is used, it is intended to include “heteroaryl” (whichwill be defined below).

Examples of heterocycles include, but are not limited to, acridinyl,azetidinyl, azocinyl, benzimidazolyl, benzofuranyl, benzothiofuranyl,benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzthiazolyl,benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl,benzimidazolinyl, carbazolyl, 4aH-carbazolyl, carbolinyl, chromanyl,chromenyl, cinnolinyl, decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl,dihydrofuro[2,3-b]tetrahydrofuran, furyl, furazanyl, imidazolidinyl,imidazolinyl, imidazolyl, 1H-indazolyl, imidazolopyridinyl, indolenyl,indolinyl, indolizinyl, indolyl (e.g., 1H-indolyl), isatinoyl,isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl, isoindolyl,isoquinolinyl, isothiazolyl, isothiazolopyridinyl, isoxazolyl,isoxazolopyridinyl, methylenedioxyphenyl, morpholinyl, naphthyridinyl,octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl,1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl,oxazolyl, oxazolopyridinyl, oxazolidinylperimidinyl, oxindolyl,pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazinyl,phenothiazinyl, phenoxathiinyl, phenoxazinyl, phthalazinyl, piperazinyl,homopiperazinyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl,pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl,pyrazolopyridinyl, pyrazolyl, pyridazinyl, pyridooxazolyl,pyridoimidazolyl, pyridothiazolyl, pyridinyl, pyrimidinyl, pyrrolidinyl,pyrrolinyl, 2-pyrrolidonyl, 2H-pyrrolyl, pyrrolyl, quinazolinyl,quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl, tetrazolyl,tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl,6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl,1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, thianthrenyl, thiazolyl,thienyl, thiazolopyridinyl, thienothiazolyl, thienooxazolyl,thienoimidazolyl, thiophenyl, triazinyl, 1,2,3-triazolyl,1,2,4-triazolyl, 1,2,5-triazolyl, 1,3,4-triazolyl, and xanthenyl. Alsoincluded are fused ring and spiro compounds containing, for example, theabove heterocycles. Examples of 5- to 10-membered heterocycles include,but are not limited to, pyridinyl, thienyl, pyrrolyl, furyl, pyrazolyl,pyrazinyl, piperazinyl, piperidinyl, imidazolyl, imidazolidinyl,indolyl, tetrazolyl, isoxazolyl, morpholinyl, oxazolyl, oxadiazolyl,oxazolidinyl, tetrahydrofuranyl, thiadiazinyl, thiadiazolyl, thiazolyl,triazinyl, triazolyl, benzimidazolyl, IH-indazolyl, benzofuranyl,benzothiofuranyl, benztetrazolyl, benzotriazolyl, benzisoxazolyl,benzoxazolyl, oxindolyl, benzoxazolinyl, benzthiazolyl,benzisothiazolyl, isatinoyl, isoquinolinyl, octahydroisoquinolinyl,tetrahydroisoquinolinyl, tetrahydroquinolinyl, isoxazolopyridinyl,quinazolinyl, quinolinyl, isothiazolopyridinyl, thiazolopyridinyl,oxazolopyridinyl, imidazolopyridinyl, and pyrazolopyridinyl. Examples of5- to 6-membered heterocycles include, but are not limited to,pyridinyl, furyl, thienyl, pyrrolyl, pyrazolyl, pyrazinyl, piperazinyl,piperidinyl, imidazolyl, imidazolidinyl, indolyl, tetrazolyl,isoxazolyl, mor-pholinyl, oxazolyl, oxadiazolyl, oxazolidinyl,tetrahydrofuranyl, thiadiazinyl, thiadiazolyl, thiazolyl, triazinyl, andtriazolyl. Also included are fused ring and spiro compounds containing,for example, the above heterocycles. Examples of a bicyclic heterocyclicgroup include, but are not limited to, quinolinyl, isoquinolinyl,phthalazinyl, quinazolinyl, indolyl, isoindolyl, indolinyl,1H-indazolyl, benzimidazolyl, 1,2,3,4-tetrahydroquinolinyl,1,2,3,4-tetrahydroisoquinolinyl, 5,6,7,8-tetrahydro-quinolinyl,2,3-dihydrobenzofuranyl, chromanyl, 1,2,3,4-tetrahydroquinoxalinyl, and1,2,3,4-tetrahydro-quinazolinyl.

The term “heteroaryl” is intended to mean stable monocyclic andpolycyclic aromatic hydrocarbons that include at least one heteroatomring member such as sulfur, oxygen, or nitrogen. Heteroaryl groups areheterocyclyl groups which are aromatic, and may include, withoutlimitation, pyridyl, pyrrolyl, pyrimidinyl, pyrazinyl, pyridazinyl,triazinyl, furyl, quinolyl, isoquinolyl, thienyl, imidazolyl, thiazolyl,indolyl (e.g., 1H-indolyl), pyrroyl, oxazolyl, benzofuryl, benzothienyl,benzthiazolyl, isoxazolyl, pyrazolyl, triazolyl, tetrazolyl, indazolyl(e.g., 1H-indazolyl), 1,2,4-thiadiazolyl, isothiazolyl, purinyl,carbazolyl, benzimidazolyl, indolinyl, benzodioxolanyl, andbenzodioxane. Heteroaryl groups may be substituted or unsubstituted. Thenitrogen atom may be substituted or unsubstituted (i.e., N or NR whereinR is H or another substituent, if defined). The nitrogen and sulfurheteroatoms may optionally be oxidized (i.e., N→0 and S(O)_(p), whereinp is 0, 1 or 2).

The term “halo” or “halogen” includes fluoro, chloro, bromo and iodo.

As used herein, the term “substituted” refers to at least one hydrogenatom that is replaced with a non-hydrogen group, provided that normalvalencies are maintained and that the substitution results in a stablecompound. When a group is noted as “optionally substituted”, the groupmay or may not contain non-hydrogen substituents. When present, thesubstituent(s) may be selected from alkyl, halo (e.g., chloro, bromo,iodo, fluoro), hydroxyl, alkoxy, oxo, alkanoyl, aryloxy, alkanoyloxy,amino (—NH₂), alkylamino (—NHalkyl), cycloalkylamino (—NHcycloalkyl),arylamino (—NHaryl), arylalkylamino (—NHarylalkyl), disubstituted amino(e.g., in which the two amino substituents are selected from alkyl, arylor arylalkyl, including substituted variants thereof, with specificmention being made to dimethylamino), alkanoylamino, aroylamino,arylalkanoylamino, thiol, alkylthio, arylthio, arylalkylthio,alkylthiono, arylthiono, arylalkylthiono, alkylsulfonyl, arylsulfonyl,arylalkylsulfonyl, sulfonamide (e.g., —SO₂NH₂), substituted sulfonamide(e.g., —SO₂NHalkyl, —SO₂NHaryl, —SO₂NHarylalkyl, or cases where thereare two substituents on one nitrogen selected from alkyl, aryl, oralkylalkyl), nitro, cyano, carboxy, unsubstituted amide (i.e. —CONH₂),substituted amide (e.g., —CONHalkyl, —CONHaryl, —CONHarylalkyl or caseswhere there are two substituents on one nitrogen selected from alkyl,aryl, or alkylalkyl), alkoxycarbonyl, aryl, guanidine, heterocyclyl(e.g., pyridyl, furyl, morpholinyl, pyrrolidinyl, piperazinyl, indolyl,imidazolyl, thienyl, thiazolyl, pyrrolidyl, pyrimidyl, piperidinyl,homopiperazinyl), and mixtures thereof. The substituents may themselvesbe optionally substituted, and may be either unprotected, or protectedas necessary, as known to those skilled in the art, for example, astaught in Greene, et al., “Protective Groups in Organic Synthesis”, JohnWiley and Sons, Second Edition, 1991, hereby incorporated by referencein its entirety.

In cases wherein there are nitrogen atoms (e.g., amines) oncompounds/complexes of the present disclosure, these may be converted toN-oxides by treatment with an oxidizing agent (e.g., mCPBA and/orhydrogen peroxides) to afford other compounds of this disclosure. Thus,shown and claimed nitrogen atoms are considered to cover both the shownnitrogen and its N-oxide (NO) derivative.

Gold(III) Complexes

The present disclosure provides mononuclear and binuclear gold(III)complexes having medicinal or pharmaceutical properties, preferablyantitumor or anticancer properties. In these gold(III) complexes, eachgold(III) atom is coordinated in a mixed ligand environment, preferablycoordinated by (i) a dithiocarbamate ligand and (ii) either abipyrimidine (e.g., a 2,2′-bipyrimidine) ligand or a bipyridine (e.g., a2,2′-bypyridine) ligand. The generic structures of a dithiocarbamate, a2,2′-bipyrimidine, and a 2,2′-bipyridine are shown below:

The coordination of each of the dithiocarbamate, bipyrimidine, andbipyridine ligand to a gold(III) atom is preferably in a bidentatemanner. In some embodiments, the gold(III) complex is binuclear (i.e.,contains two gold(III) atoms), with a central bipyrimidine ligand thatbridges the two gold(III) atoms (each gold(III) atom being coordinatedin a bidentate manner to the central bipyrimidine ligand), anddithiocarbamate ligands that coordinate in a bidentate fashion to eachof the gold(III) atoms. In some embodiments, the gold(III) complex ismononuclear (i.e., contains one gold(III) atom), with a singledithiocarbamate ligand and a single bipyridine ligand coordinated to thegold(III) atom in a bidentate fashion.

Gold(III) Complex of Formula (I)

In a first aspect, the present disclosure provides a gold(III) complexof formula (I),

or a pharmaceutically acceptable salt, solvate, tautomer, orstereoisomer thereof,

wherein:

R¹ and R² are each independently a hydrogen, an optionally substitutedalkyl, an optionally substituted cycloalkyl, an optionally substitutedarylalkyl, or an optionally substituted aryl;

R³ and R⁴ are each independently a hydrogen, an optionally substitutedalkyl, an optionally substituted cycloalkyl, an optionally substitutedarylalkyl, an optionally substituted aryl, an optionally substitutedheterocyclyl, an optionally substituted alkoxy, a hydroxyl, a halo, anitro, a cyano, a N-monosubstituted amino group, or a N,N-disubstitutedamino group; and

X is Cl, Br, or I.

In terms of R¹ and R², these substituents may be the same or different.Preferably R¹ and R² are the same. In some embodiments, R¹ and R² areeach independently an optionally substituted alkyl or an optionallysubstituted arylalkyl. Preferably, R¹ is a C₁ to C₈ alkyl, preferably aC₂ to C₇ alkyl, preferably a C₃ to C₆ alkyl, or a C₇ to C₁₂ arylalkyl,preferably a C₈ to C₁₁ arylalkyl, preferably a C₉ to C₁₀ arylalkyl. Inpreferred embodiments, R¹ and R² are the same, and are each methyl,ethyl, or benzyl, most preferably methyl or ethyl.

In terms of R³ and R⁴, these substituents may be the same or different.Preferably R³ and R⁴ are the same. R³ and R⁴ may be, independently,located at a position ortho to a nitrogen atom of the bipyrimidine ring,or may be located at a position meta to both nitrogen atoms of thebipyrimidine ring. In some embodiments, R³ and R⁴ are each hydrogen, anoptionally substituted alkyl (preferably a C₁ to C₈ alkyl, preferably aC₂ to C₇ alkyl, preferably a C₃ to C₆ alkyl), an optionally substitutedalkoxy (preferably a C₁ to C₄ alkoxy, preferably a C₂ to C₃ alkoxy), ahydroxyl, a N-monosubstituted amino group (preferably an optionallysubstituted alkylamino or an optionally substituted aryalkylamino, e.g.,methylamino or benzylamino), or a N,N-disubstituted amino group(preferably an amino group having two substituents selected from anoptionally substituted alkyl and an optionally substituted arylalkyl,e.g., dimethylamino). In preferred embodiments, R³ and R⁴ are the same,and are each hydrogen.

In the gold(III) complex of formula (I), X represents a counteranionwhich is outside of the coordination sphere of the complex, i.e., notdirectly bound to the gold atom. As the gold(III) complex of formula (I)is binuclear (contains two gold atoms in the +3 oxidation state), withtwo total anionic ligands (each dithiocarbamate ligand carries a −1charge), the gold(III) complex of formula (I) includes four X⁻counteranions. In preferred embodiments, X is Cl and/or Br, preferably Xis Cl.

In preferred embodiments, the gold(III) complex of formula (I) isselected from the group consisting of

In the most preferred embodiments, the gold(III) complex of formula (I)is

Gold(III) complex of formula (II)

According to a second aspect, the present disclosure also provides agold(III) complex of formula (II),

or a pharmaceutically acceptable salt, solvate, tautomer, orstereoisomer thereof,

wherein:

R¹ and R² are each independently a hydrogen, an optionally substitutedalkyl, an optionally substituted cycloalkyl, an optionally substitutedarylalkyl, or an optionally substituted aryl;

R³ and R⁴ are each independently an optionally substituted alkyl, anoptionally substituted cycloalkyl, an optionally substituted arylalkyl,an optionally substituted aryl, an optionally substituted heterocyclyl,an optionally substituted alkoxy, a hydroxyl, a halo, a nitro, a cyano,a N-monosubstituted amino group, or a N,N-disubstituted amino group; andX is Cl, Br, or I.

In terms of R¹ and R², these substituents may be the same or different.Preferably R¹ and R² are the same. In some embodiments, R¹ and R² areeach independently an optionally substituted alkyl or an optionallysubstituted arylalkyl. Preferably, R¹ is a C₁ to C₈ alkyl, preferably aC₂ to C₇ alkyl, preferably a C₃ to C₆ alkyl, or a C₇ to C₁₂ arylalkyl,preferably a C₈ to C₁₁ arylalkyl, preferably a C₉ to C₁₀ arylalkyl. Inpreferred embodiments, R¹ and R² are the same, and are each methyl,ethyl, or benzyl, most preferably methyl or ethyl.

In terms of R³ and R⁴, these substituents may be the same or different.Preferably R³ and R⁴ are the same. In some embodiments, R³ and R⁴ areeach hydrogen, an optionally substituted alkyl (preferably a C₁ to C₈alkyl, preferably a C₂ to C₇ alkyl, preferably a C₃ to C₆ alkyl), anoptionally substituted alkoxy (preferably a C₁ to C₄ alkoxy, preferablya C₂ to C₃ alkoxy), a hydroxyl, a N-monosubstituted amino group(preferably an optionally substituted alkylamino or an optionallysubstittued aryalkylamino, e.g., methylamino or benzylamino), or aN,N-disubstituted amino group (preferably an amino group having twosubstituents selected from an optionally substituted alkyl and anoptionally substituted arylalkyl, e.g., dimethylamino). In preferredembodiments, R³ and R⁴ are the same, and are each hydroxyl.

In the gold(III) complex of formula (II), X represents a counteranionwhich is outside of the coordination sphere of the complex, i.e., notdirectly bound to the gold atom. As the gold(III) complex of formula(II) is mononuclear (contains one gold atom in the +3 oxidation state),with one total anionic ligand (the diothiocarbamate ligand) carrying a−1 charge, the gold(III) complex of formula (II) includes two X⁻counteranions. In preferred embodiments, X is Cl and/or Br, preferably Xis Cl.

Methods of Making

The “gold(III) complexes” (those of formula (I) and/or formula (II)) ofthe present disclosure may be prepared by any complexation method knownto those of ordinary skill in the art. The following methods set forthbelow are provided for illustrative purposes and are not intended tolimit the scope of the disclosure.

The gold(III) complexes may, for example, be synthesized according to astepwise complexation route. Briefly, the gold(III) complexes may beformed by mixing together an aqueous solution of a gold(III) salt with aligand solution of either a bipyrimidine ligand of formula (III) or abipyridine ligand of formula (IV) to form a gold-ligand mixture.

wherein R³and R⁴ are as defined previously.

Exemplary gold(III) salts include, but are not limited to, sodiumtetrachloroaurate(III), potassium tetrachloroaurate(III), cesiumtetrachloroaurate(III), sodium tetrabromoaurate(III), potassiumtetrabromoaurate(III), cesium tetrabromoaurate(III), as well as mixturesor hydrates thereof. Typically, a concentration of the gold(III) salt inthe aqueous solution may range from 0.1 to 1.5 mM, preferably 0.2 to 1.4mM, preferably 0.3 to 1.3 mM, preferably 0.4 to 1.2 mM, preferably 0.5to 1.0 mM.

The ligand solution may be formed with one or more organic solvents,including, but not limited to, aromatic solvents (e.g., benzene,ethylbenzene, o-xylene, m-xylene, p-xylene, and mixtures of xylenes,toluene, mesitylene, anisole, 1,2-dimethoxybenzene,α,α,α-trifluoromethylbenzene, fluorobenzene, heavy aromatic naptha),alkane solvents (e.g., pentane, cyclopentane, hexanes, cyclohexane,heptanes, cycloheptane, octanes), ethers (e.g. diethyl ether,tetrahydrofuran, 1,4-dioxane, tetrahydropyran, t-butyl methyl ether,cyclopentyl methyl ether, di-isopropyl ether), glycol ethers (e.g.1,2-dimethoxyethane, diglyme, triglyme), chlorinated solvents (e.g.chlorobenzene, dichloromethane, 1,2-dichloroethane, 1,1-dichloroethane,chloroform, carbon tetrachloride), ester solvents (e.g. ethyl acetate,propyl acetate), ketones (e.g. acetone, butanone), formamides/acetamides(e.g., formamide, dimethyl formamide, dimethyl acetamide), monoalcohols(e.g., methanol, ethanol, propanol, isopropanol, n-butanol, isobutanol,n-pentanol, n-hexanol, terpineol, menthol, prenol,3-methyl-3-buten-1-ol, 2-ethyl-1-hexanol, 2-ethyl-1-butanol,2-propylheptan-1-ol, 2-butyl-1-octanol, benzyl alcohol), polyalcoholsincluding glycols (e.g., ethylene glycol, diethylene glycol, triethyleneglycol, tetraethylene glycol, propylene glycol, dipropylene glycol,1,3-propanediol, 1,3-butanediol, 1,4-butanediol, 1,6-hexanediol,glycerol, pentaerythritol, manitol, sorbitol), as well as mixturesthereof. Preferably a mixture of a monoalcohol (e.g., ethanol) and achlorinated solvent (e.g., dichloromethane) is used as the ligandsolution solvent, for example at a vol:vol ratio of 1:1 to 5:1,preferably 2:1 to 4:1, preferably 3:1. Typically, a concentration of thebipyrimidine ligand of formula (III) or the bipyridine ligand of formula(IV) in the ligand in the ligand solution may range from 0.1 to 1.5 mM,preferably 0.2 to 1.4 mM, preferably 0.3 to 1.3 mM, preferably 0.4 to1.2 mM, preferably 0.5 to 1.0 mM.

The aqueous solution of the gold(III) complex and the ligand solutionmay be mixed together as is, or alternatively may be added to amonoalcohol solvent (e.g., ethanol) to form the gold-ligand mixture.When making a binuclear complex (i.e., the gold(III) complex of formulaI), a mole ratio of the gold(III) salt to the bipyrimidine ligand offormula (III) typically ranges from 1.7:1 to 2.3:1, preferably 1.8:1 to2.2:1, preferably 1.9:1 to 2.1:1, preferably 2:1. When making amononuclear complex (i.e., the gold(III) complex of formula II), a moleratio of the gold(III) salt to the bipyridine ligand of formula (IV)typically ranges from 0.7:1 to 1.3:1, preferably 0.8:1 to 1.2:1,preferably 0.9:1 to 1.1:1, preferably 1:1. The gold-ligand mixture maybe agitated (e.g., using an agitator, a vortexer, a rotary shaker, amagnetic stirrer, a centrifugal mixer, an overhead stirrer, etc.) forany amount of time sufficient for complexation, typically from 0.5 to 10hours, preferably 1 to 6 hours, preferably 2 to 3 hours.

A dithiocarbamate salt solution may then be mixed with the gold-ligandmixture to form a reaction mixture. The dithiocarbamate salt solutioncontains a dithiocarbamate salt in water or an alcohol solvent,preferably ethanol. Typically, a concentration of dithiocarbamate saltin the dithiocarbamate salt solution may range from 0.1 to 1.5 mM,preferably 0.2 to 1.4 mM, preferably 0.3 to 1.3 mM, preferably 0.4 to1.2 mM, preferably 0.5 to 1.0 mM. A mole ratio of the dithiocarbamatesalt to the gold(III) salt above used to form the gold-ligand mixturemay be in a range of 0.7:1 to 1.3:1, preferably 0.8:1 to 1.2:1,preferably 0.9:1 to 1.1:1, preferably 1:1. The reaction mixture may beagitated for 0.1 to 10 hours, 0.5 to 6 hours, or 1 to 3 hours.

The dithiocarbamate salt is preferably represented by formula (V)

where R¹ and R² are as defined above, and M⁺ is an alkali metal cation(e.g. sodium, potassium, cesium, lithium, and rubidium), ammonium, anoptionally substituted alkylammonium, an optionally substitutedarylammonium, or an optionally substituted alkylarylammonium. Exemplarydithiocarbamate salts include, but are not limited to, sodiumdimethyldithiocarbamate, potassium dimethyldithiocarbamate, ammoniumdimethyldithiocarbamate, sodium diethyldithiocarbamate, potassiumdiethyldithiocarbamate, ammonium diethyldithiocarbamate, sodiumdibenzyldithiocarbamate, potassium dibenzyldithiocarbamate, ammoniumdibenzyldithiocarbamate, and hydrates thereof.

The progress of any such reactions may be monitored by methods known tothose of ordinary skill in the art, such as thin layer chromatography,gas chromatography, nuclear magnetic resonance, infrared spectroscopy,ultraviolet detection, or mass spectroscopy.Precipitation/crystallization of the gold(III) complexes may occurfollowing the above procedures and the gold(III) complexes may beisolated and purified by methods known to those of ordinary skill in theart, such as one or more of crystallization, precipitation, filtration,solvent evaporation, drying, and the like.

Of course, it should be understood that the gold(III) complexes may besynthesized through various other synthetic schemes, reactions types andconditions, and isolation/purification procedures and still beconsidered a part of the present disclosure.

Pharmaceutical Compositions

The present disclosure relates to a pharmaceutical composition whichcomprises a therapeutically effective amount of one or more of thegold(III) complexes, formulated together with one or morepharmaceutically acceptable carriers and/or excipients, and optionally,one or more additional therapeutic agents. As described in detail below,the pharmaceutical compositions of the present disclosure may bespecially formulated for administration in solid or liquid form,including those adapted for the following: (1) oral administration, forexample, drenches (aqueous or non-aqueous solutions or suspensions),tablets, e.g., those targeted for buccal, sublingual, and systemicabsorption, boluses, powders, granules, pastes for application to thetongue; (2) parenteral administration, for example, by subcutaneous,intramuscular, intravenous, epidural injection, or intratumoral, as, forexample, a sterile solution or suspension, or sustained releaseformulation; (3) topical application, for example, as a cream, ointment,or a controlled release patch or spray applied to the skin; (4)intravaginally or intrarectally, for example, as a pessary, cream orfoam; (5) sublingually; (6) ocularly; (7) transdermally; or (8) nasally.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds/complexes, materials, compositions, and/or dosage formswhich are, within the scope of sound medical judgment, suitable for usein contact with the tissues of human beings and animals withoutexcessive toxicity, irritation, allergic response, or other problem orcomplication, commensurate with a reasonable benefit/risk ratio.

As used herein, a “composition” or a “pharmaceutical composition” refersto a mixture of an active ingredient(s) with other chemical components,such as pharmaceutically acceptable carriers and/or excipients. Onepurpose of a composition is to facilitate administration of thegold(III) complexes disclosed herein in any of their embodiments to asubject. Pharmaceutical compositions of the present disclosure may bemanufactured by processes well known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping or lyophilizing processes.Depending on the intended mode of administration (e.g., oral,parenteral, or topical), the composition can be in the form of solid,semi-solid or liquid dosage forms, such as tablets, suppositories,pills, capsules, powders, liquids, or suspensions, preferably in unitdosage form suitable for single administration of a precise dosage.

The term “active ingredient” or “active compound”, as used herein,refers to an ingredient in the composition that is biologically active,for example, one or more of the gold(III) complexes. In someembodiments, additional therapeutic agents, in addition to the gold(III)complexes of the current disclosure, may be incorporated into apharmaceutical composition, for example, a second active ingredientwhich is chemically distinct from the gold(III) complexes.

When the gold(III) complexes are administered as pharmaceuticals, tohumans and animals, they can be given per se or as a pharmaceuticalcomposition containing the active ingredient(s) in combination with apharmaceutically acceptable carrier and/or excipient. The pharmaceuticalcomposition may contain, for example, up to 99.9 wt. %, preferably up to99 wt. %, preferably up to 90 wt. %, preferably up to 80 wt. %,preferably up to 70 wt. %, preferably up to 60 wt. %, preferably up to50 wt. %, preferably up to 40 wt. %, preferably up to 30 wt. %,preferably up to 20 wt. %, preferably up to 10 wt. %, preferably up to 5wt. %, preferably up to 1 wt. %, preferably up to 0.5 wt. %, preferablyup to 0.1 wt. %, preferably up to 0.01 wt. %, preferably up to 0.001 wt.%, preferably up to 0.0001 wt. %, of the gold(III) complex, based on atotal weight of the pharmaceutical composition. For example, whenformulated as a solution, the pharmaceutical composition may contain 1to 50 μM, preferably 2 to 45 μM, preferably 3 to 40 μM, preferably 4 to35 μM, preferably 5 to 30 μM, preferably 6 to 25 μM, preferably 7 to 20μM, preferably 8 to 15 μM, preferably 9 to 12 μM, preferably 10 to 11 μMof the gold(III) complex relative to a total volume of thepharmaceutical composition.

In some embodiments, the active ingredient of the current disclosure,e.g., the gold(III) complexes, a salt thereof, a solvate thereof, atautomer thereof, a stereoisomer thereof, or any mixtures thereof, mayprovide utility as an anticancer agent in reducing the viability ofcancer cells derived from human cancer cell lines including, but notlimited to, breast cancer cell lines (e.g., MDA-MB-231, MCF-7, SK-BR-3,T47D, VP303); stomach cancer cell lines (e.g., N87, SNU-16, SNU-5,SNU-1, KATO III, AGS); colon/colorectal cancer cell lines (e.g.,HCT-116, CACO-2, HT-29, HCT15, MDST8, GPSd, DLD1, SW620, SW403, T84);leukemia cell lines (e.g., HL-60, CESS, CCRF-CEM, CEM/C1, KASUMI-1,ARH-77); liver cancer cell lines (e.g., HepG2, PLC/PRF/5, THLE-3, C3A,SNU-182, SNU-398, SNU-387, SNU-423, SNU-475, SNU-449, and Hep 3B2.1-7);lung cancer cell lines (e.g., A549, NCI-H460, SHP-77, COR-L23/R,NCI-H69/LX20); brain tumor cell lines (e.g., U251); ovarian cancer celllines (e.g., NCI-ADR/RES, OVCAR-03, A2780, A2780cis, OV7, PEO23);prostate cancer cell lines (e.g., DU145, PC-3); renal cancer cell lines(e.g., 786-0); skin cancer or melanoma cell lines (e.g., UACC-62, C32TG,A375, MCC26), bone cancers such as osteosarcoma cell lines (e.g.,MG-63), and cervical cancer cell lines (e.g., ME-180, R-ME-180).Preferably, the active ingredient of the current disclosure, e.g., thegold(III) complexes, a salt thereof, a solvate thereof, a tautomerthereof, a stereoisomer thereof, or any mixtures thereof, providesutility as an anticancer agent in reducing the viability of cancer cellsderived from bone cancers such as osteosarcoma cell lines (e.g., MG-63),lung cancer cell lines (e.g., A549, NCI-H460, SHP-77, COR-L23/R,NCI-H69/LX20), prostate cancer cell lines (e.g., DU145, PC-3), breastcancer cell lines (e.g., MDA-MB-231, MCF-7, SK-BR-3, T47D, VP303),ovarian cancer cell lines (e.g., NCI-ADR/RES, OVCAR-03, A2780, A2780cis,OV7, PEO23), and cervical cancer cell lines (e.g., ME-180, R-ME-180).

In preferred embodiments, the active ingredient of the currentdisclosure, e.g., the gold(III) complexes, a salt thereof, a solvatethereof, a tautomer thereof, a stereoisomer thereof, or any mixturesthereof, may provide utility as an anticancer agent in reducing theviability of cancer cells derived from human cancer cell lines which areresistant to, or which are susceptible to becoming resistant to, othertherapeutic agents/chemotherapy agents such as cisplatin anddoxorubicin, with specific mention being made to cisplatin anddoxorubicin resistant ovarian cancers (e.g., A2780cis) and cisplatinresistant cervical cancers (e.g., R-ME-180).

In some embodiments, the cancer cells are collected from a human patientwho is at risk of having, is suspected of having, has been diagnosedwith, or is being monitored for recurrence of at least one type ofcancer, preferably at least one of bone cancer, lung cancer, prostatecancer, breast cancer, ovarian cancer, and cervical cancer.

In some embodiments, the ability of the active ingredient to reduce theviability of cancer cells may be determined by contacting thepharmaceutical composition with the cancer cells and then performingcell viability assays. Methods of such assays include, but are notlimited to, sulforhodamine-B (SRB) assay, ATP test, Calcein AM assay,clonogenic assay, ethidium homodimer assay, Evans blue assay,2′,7′-dichlorofluorescin diacetate (DCFDA) or2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) staining assay,fluorescein diacetate hydrolysis/propidium iodide staining assay,annexin V/fluorescein isothiocyanate (FITC)/propidium iodide stainingassay, flow cytometry, Formazan-based assays (MTT, XTT), greenfluorescent protein assay, lactate dehydrogenase (LDH) assay, methylviolet assay, propidium iodide assay, Resazurin assay, trypan blueassay, 4′,6′-diamidino-2-phenylindole (DAPI) assay, TUNEL assay, afluorochrome-labeled inhibitors of caspases (FLICA)-based assay, primary(1°) colonosphere formation assay, thioredoxin reductase assay, 20Sproteasome activity assay, in vitro scratch assay (for cell migrationanalysis).

As is well understood in the art, the IC₅₀ value of a compound/mixtureis a concentration of that compound/mixture which causes the death of50% of the cellular population to which the compound/mixture is added.In some embodiments, the IC₅₀ of the gold(III) complexes, the saltthereof, the solvate thereof, the tautomer thereof, the stereoisomerthereof, or mixtures thereof against bone cancer, lung cancer, prostatecancer, breast cancer, ovarian cancer, and cervical cancer cells, isless than 200 μM, preferably less than 150 μM, preferably less than 100μM, preferably less than 90 μM, preferably less than 80 μM, preferablyless than 70 μM, preferably less than 60 μM, preferably less than 50 μM,preferably less than 40 μM, preferably less than 30 μM, preferably lessthan 25 μM, preferably less than 20 μM, preferably less than 15 μM,preferably less than 10 μM, preferably less than 5 μM, preferably lessthan 4 μM, preferably less than 3 μM, preferably less than 2 μM,preferably less than 1 μM, for example, from 0.5 to 25 μM, preferablyfrom 0.6 to 20 μM, preferably from 0.7 to 15 μM, preferably from 0.8 to10 μM, preferably from 0.9 to 9 μM, preferably from 1 to 8 μM,preferably from 1.1 to 7 μM, preferably from 1.2 to 6 μM, preferablyfrom 1.3 to 5 μM, preferably from 1.4 to 4 μM, preferably from 1.5 to 3μM, preferably from 1.6 to 2 μM.

In some embodiments, additional therapeutic agents in addition to thegold(III) complexes of the current disclosure may be incorporated intothe pharmaceutical composition.

In some embodiments, the pharmaceutical composition includes anadditional therapeutic agent that is chemically distinct from thegold(III) complex (of formula (I) or formula (II)), such as achemotherapeutic agent or an anticancer agent, for the treatment orprevention of neoplasm, of tumor or cancer cell division, growth,proliferation and/or metastasis in the subject; induction of death orapoptosis of tumor and/or cancer cells; and/or any other forms ofproliferative disorder.

The additional therapeutic agent may be an anticancer agent and mayinclude, but is not limited to, at least one of a mitotic inhibitor; analkylating agent; an antimetabolite; a cell cycle inhibitor; an enzyme;a topoisomerase inhibitor (e.g., doxorubicin); a biological responsemodifier; an anti-hormone; a tubulin inhibitor; a tyrosine-kinaseinhibitor; an antiangiogenic agent such as MMP-2, MMP-9 and COX-2inhibitor; an anti-androgen; a platinum coordination complex (cisplatin,oxaliplatin, carboplatin); a substituted urea such as hydroxyurea; amethylhydrazine derivative; an adrenocortical suppressant, e.g.,mitotane, aminoglutethimide; a hormone and/or hormone antagonist such asthe adrenocorticosteriods (e.g., prednisone), progestins (e.g.,hydroxyprogesterone caproate), an estrogen (e.g., diethylstilbestrol);an antiestrogen such as tamoxifen; androgen, e.g., testosteronepropionate; and an aromatase inhibitor, such as anastrozole, andAROMASIN (exemestane).

Exemplary additional therapeutic agents include, but are not limited to,tubulin binding agents including paclitaxel, epothilone, docetaxel,discodermolide, etoposide, vinblastine, vincristine, teniposide,vinorelbine, and vindesine; tyrosine-kinase inhibitors includingimatinib, nilotinib, dasatinib, bosutinib, ponatinib, and bafetinib;alkylating antineoplastic agents including busulfan, carmustine,chlorambucil, cyclophosphamide, cyclophosphamide, dacarbazine,ifosfamide, lomustine, mechlorethamine, melphalan, mercaptopurine,procarbazine; antimetabolites including cladribine, cytarabine,fludarabine, gemcitabine, pentostatin, 5-fluorouracil, clofarabine,capecitabine, methotrexate, thioguanine; cytotoxic antibiotics includingdaunorubicin, idarubicin, mitomycin, actinomycin, epirubicin;topoisomerase inhibitors including doxorubicin, irinotecan,mitoxantrone, topotecan, and mixtures thereof.

As used herein, the phrase “pharmaceutically acceptable carrier and/orexcipient” means a pharmaceutically acceptable material, composition orvehicle, such as a liquid or solid filler, carrier, diluent, excipient,manufacturing aid (e.g., lubricant, talc magnesium, calcium or zincstearate, or steric acid), or solvent encapsulating material, involvedin carrying or transporting the subject compound from one organ, orportion of the body, to another organ, or portion of the body. Eachcarrier must be “acceptable” in the sense of being compatible with theother ingredients of the formulation and not injurious to the patient.Some examples of materials which can serve as pharmaceuticallyacceptable carriers include: (1) sugars, such as lactose, glucose andsucrose; (2) starches, such as corn starch and potato starch; (3)cellulose, and its derivatives, such as sodium carboxymethyl cellulose,ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5)malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter andsuppository waxes; (9) oils, such as peanut oil, cottonseed oil,safflower oil, sesame oil, olive oil, corn oil, castor oil, and soybeanoil; (10) glycols, such as propylene glycol; (11) polyols, such asglycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, suchas ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents,such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid;(16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution;(19) ethyl alcohol and/or other organic solvents (e.g., DMSO); (20) pHbuffered solutions; (21) polyesters, polycarbonates and/orpolyanhydrides; and/or (22) other non-toxic compatible substancesemployed in pharmaceutical formulations, such as cyclodextrins,liposomes, micelle forming agents, e.g., bile acids, polyethoxylatedoils (e.g., polyethoxylated castor oil) just to name a few.

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the pharmaceuticalcompositions.

Examples of pharmaceutically-acceptable antioxidants include: (1) watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Methods of preparing these pharmaceutical compositions include the stepof bringing into association the gold(III) complex with thepharmaceutically acceptable carrier and/or excipient, and, optionally,one or more accessory ingredients. In general, the compositions areprepared by uniformly and intimately bringing into association agold(III) complex of the present disclosure with liquid carriers, orfinely divided solid carriers, or both, and then, if necessary, shapingthe product.

Pharmaceutical compositions of the present disclosure suitable for oraladministration may be in the form of capsules, cachets, pills, tablets,lozenges (using a flavored basis, usually sucrose and acacia ortragacanth), powders, granules, or as a solution or a suspension in anaqueous or non-aqueous liquid, or as an oil-in-water or water-in-oilliquid emulsion, or as an elixir or syrup, or as pastilles (using aninert base, such as gelatin and glycerin, or sucrose and acacia) and/oras mouth washes and the like, each containing a predetermined amount ofa gold(III) complex as an active ingredient. A gold(III) complex of thepresent disclosure may also be administered as a bolus, electuary orpaste.

In solid dosage forms of the present disclosure for oral administration(capsules, tablets, pills, dragees, powders, granules, troches and thelike), the active ingredient is mixed with one or more pharmaceuticallyacceptable carriers and/or excipients, such as sodium citrate ordicalcium phosphate, and/or any of the following: (1) fillers orextenders, such as starches, lactose, sucrose, glucose, mannitol, and/orsilicic acid; (2) binders, such as, for example, carboxymethylcellulose,alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3)humectants, such as glycerol; (4) disintegrating agents, such asagar-agar, calcium carbonate, potato or tapioca starch, alginic acid,certain silicates, and sodium carbonate; (5) solution retarding agents,such as paraffin; (6) absorption accelerators, such as quaternaryammonium compounds and surfactants, such as poloxamer and sodium laurylsulfate; (7) wetting agents, such as, for example, cetyl alcohol,glycerol monostearate, and non-ionic surfactants (e.g., fatty acidesters of sorbitan and polyalkolyated fatty acid esters of sorbitan suchas TWEEN 80, available from Sigma-Aldrich); (8) absorbents, such askaolin and bentonite clay; (9) lubricants, such as talc, calciumstearate, magnesium stearate, solid polyethylene glycols, sodium laurylsulfate, zinc stearate, sodium stearate, stearic acid, and mixturesthereof; (10) coloring agents; and (11) controlled release agents suchas crospovidone or ethyl cellulose. In the case of capsules, tablets andpills, the pharmaceutical compositions may also comprise bufferingagents. Solid compositions of a similar type may also be employed asfillers in soft and hard shelled gelatin capsules using such excipientsas lactose or milk sugars, as well as high molecular weight polyethyleneglycols and the like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent. The tablets, and other soliddosage forms of the pharmaceutical compositions of the presentdisclosure may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be formulated for rapid release,e.g., freeze-dried. They may be sterilized by, for example, filtrationthrough a bacteria retaining filter, or by incorporating sterilizingagents in the form of sterile solid compositions which can be dissolvedin sterile water, or some other sterile injectable medium immediatelybefore use. These compositions may also optionally contain opacifyingagents and may be of a composition that they release the activeingredient(s) only, or preferentially, in a certain portion of thegastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions which can be used include polymeric substancesand waxes. The active ingredient can also be in micro-encapsulated form,if appropriate, with one or more of the above described excipients.

Liquid dosage forms for oral administration of the complexes of thedisclosure include pharmaceutically acceptable emulsions,microemulsions, solutions, suspensions, syrups and elixirs. In additionto the active ingredient, the liquid dosage forms may contain inertdiluents commonly used in the art, such as, for example, water or othersolvents, solubilizing agents and emulsifiers, such as ethyl alcohol,isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol,benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (inparticular, cottonseed, groundnut, com, germ, olive, castor and sesameoils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fattyacid esters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compound, may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters (including polyoxyethylene fatty acidesters of sorbitan, e.g., TWEEN 80), microcrystalline cellulose,aluminum metahydroxide, bentonite, agar-agar and tragacanth, andmixtures thereof. In preferred embodiments, the pharmaceuticalcomposition is in the form of a suspension, comprising, consisting of,or consisting essentially of the gold(III) complex and thepharmaceutically acceptable carrier and/or excipient, which ispreferably a suspending agent (preferably a polyoxyethylene sorbitanester, preferably a polyoxyethylene fatty acid ester of sorbitan, e.g.,TWEEN 80) in an inert diluent (preferably water). Preferably the contentof the suspending agent in the suspension ranges from 0.01 to 1 wt. %,preferably 0.05 to 0.8 wt. %, preferably 0.1 to 0.6 wt. %, preferably0.5 wt. %, based on a total weight of the suspension.

Formulations of the pharmaceutical compositions of the disclosure forrectal or vaginal administration may be presented as a suppository,which may be prepared by mixing one or more gold(III) complexes with oneor more suitable nonirritating excipients or carriers comprising, forexample, cocoa butter, polyethylene glycol, a suppository wax or asalicylate, and which is solid at room temperature, but liquid at bodytemperature and, therefore, will melt in the rectum or vaginal cavityand release the active compound(s).

Formulations of the pharmaceutical compositions which are suitable forvaginal administration also include pessaries, tampons, creams, gels,pastes, foams or spray formulations containing such carriers as areknown in the art to be appropriate.

Dosage forms for the topical or transdermal administration of agold(III) complex of this disclosure include powders, sprays, ointments,pastes, creams, lotions, gels, solutions, patches and inhalants. Theactive compound may be mixed under sterile conditions with apharmaceutically acceptable carrier, and with any preservatives,buffers, or propellants which may be required.

The ointments, pastes, creams and gels may contain, in addition to anactive gold(III) complex of this disclosure, excipients, such as animaland vegetable fats, oils, waxes, paraffins, starch, tragacanth,cellulose derivatives, polyethylene glycols, silicones, bentonites,silicic acid, talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a gold(III) complex ofthis disclosure, excipients such as lactose, talc, silicic acid,aluminum hydroxide, calcium silicates and polyamide powder, or mixturesof these substances. Sprays can additionally contain customarypropellants, such as chlorofluorohydrocarbons and volatile unsubstitutedhydrocarbons, such as butane and propane.

Transdermal patches have the added advantage of providing controlleddelivery of a gold(III) complex of the present disclosure to the body.Such dosage forms can be made by dissolving or dispersing the gold(III)complex in the proper medium. Absorption enhancers can also be used toincrease the flux of the gold(III) complex across the skin. The rate ofsuch flux can be controlled by either providing a rate controllingmembrane or dispersing the gold(III) complex in a polymer matrix or gel.

Ophthalmic formulations, eye ointments, powders, solutions and the like,are also contemplated as being within the scope of this disclosure.

Pharmaceutical compositions of this disclosure suitable for parenteraladministration comprise one or more gold(III) complexes of the presentdisclosure in combination with one or more pharmaceutically acceptablesterile isotonic aqueous or non-aqueous solutions, dispersions,suspensions or emulsions, or sterile powders which may be reconstitutedinto sterile injectable solutions or dispersions just prior to use,which may contain sugars, alcohols, antioxidants, buffers (e.g.,phosphate buffered saline, PBS), bacteriostats, solvents,polyalkoxylated oils such as polyethoxylated castor oil (e.g., CREMOPHORfrom Sigma-Aldrich), solutes which render the formulation isotonic withthe blood of the intended recipient or suspending or thickening agents.

Examples of suitable aqueous and non-aqueous carriers which may beemployed in the pharmaceutical compositions of the disclosure includewater, ethanol, DMSO, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants (e.g., TWEEN 80).

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms upon the subject gold(III) complexes may beensured by the inclusion of various antibacterial and antifungal agents,for example, paraben, chlorobutanol, phenol sorbic acid, and the like.It may also be desirable to include isotonic agents, such as sugars,sodium chloride, and the like into the pharmaceutical compositions. Inaddition, prolonged absorption of the injectable pharmaceutical form maybe brought about by the inclusion of agents which delay absorption suchas aluminum monostearate and gelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Injectable depot forms are made by forming microencapsuled matrices ofthe subject gold(III) complexes in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly (orthoesters) and poly (anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissue.

In preferred embodiments, the pharmaceutical compositions of thisdisclosure are formulated for parenteral administration, preferablyintratumoral injection, preferably intratumoral injection using apharmaceutically acceptable carrier and/or excipient made of 5 to 15vol. %, preferably 8 to 12 vol. %, preferably 10 vol. % DMSO, 15 to 25vol. %, preferably 18 to 22 vol. %, preferably 20 vol. % polyethoxylatedcastor oil (e.g., CREMOPHOR from Sigma-Aldrich), and 65 to 75 vol. %,preferably 68 to 72 vol. %, preferably 70 vol. % buffer (e.g., PBSbuffer).

In some embodiments, the pharmaceutical composition contains 1 to99.9999 wt. %, preferably 5 to 99.999 wt. %, preferably 10 to 99.99 wt.%, preferably 15 to 99 wt. %, preferably 20 to 90 wt. %, preferably 30to 85 wt. %, preferably 40 to 80 wt. %, preferably 50 to 75 wt. % of thepharmaceutically acceptable carrier and/or excipient, relative to atotal weight of the pharmaceutical composition.

Therapeutic Applications and Methods

According to another aspect, the present disclosure relates to a methodfor treating a proliferative disorder. The method involves administeringa therapeutically effective amount of one or more gold(III) complexesper se, or a pharmaceutical composition described above to a subject.

In some embodiments, the proliferative disorder is cancer. Types ofcancers that may be treated with the gold(III) complexes of thisdisclosure include, but are not limited to, brain cancers, skin cancers,bladder cancers, ovarian cancers, breast cancers, gastric cancers,pancreatic cancers, prostate cancers, colon/colorectal cancers, bloodcancers, lung cancers, cervical cancers, and bone cancers. In someembodiments, the gold(III) complexes of this disclosure can be used forthe treatment of any cancer type that fails to undergo apoptosis in apatient. This includes, but is not limited to: solid tumors, includingbut not limited to carcinomas; sarcomas including Kaposi's sarcoma andosteosarcoma; erythroblastoma; glioblastoma; meningioma; astrocytoma;melanoma; and myoblastoma. Treatment or prevention of non-solid tumorcancers, such as leukemia, is also contemplated by this invention.

Examples of such cancer types include neuroblastoma, intestine carcinomasuch as rectum carcinoma, colon carcinoma, familiar adenomatouspolyposis carcinoma and hereditary non-polyposis colorectal cancer,esophageal carcinoma, labial carcinoma, larynx carcinoma, hypopharynxcarcinoma, tong carcinoma, salivary gland carcinoma, gastric carcinoma,adenocarcinoma, medullary thyroid carcinoma, papillary thyroidcarcinoma, renal carcinoma, kidney parenchymal carcinoma, ovariancarcinoma, cervix carcinoma, uterine corpus carcinoma, endometriumcarcinoma, chorion carcinoma, pancreatic carcinoma, prostate carcinoma,testis carcinoma, breast carcinoma, urinary carcinoma, melanoma, braintumors such as glioblastoma, astrocytoma, meningioma, medulloblastomaand peripheral neuroectodermal tumors, Hodgkin lymphoma, non-Hodgkinlymphoma, Bur-kitt lymphoma, acute lymphatic leukemia (ALL), chroniclymphatic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloidleukemia (CML), adult T-cell leukemia lymphoma, diffuse laige B-celllymphoma (DLBCL), hepatocellular carcinoma, gall bladder carcinoma,bronchial carcinoma, small cell lung carcinoma, non-small cell lungcarcinoma, multiple myeloma, basalioma, teratoma, retinoblastoma,choroid melanoma, seminoma, rhabdomyosarcoma, craniopharyngioma,osteosarcoma, chondrosarcoma, myosarcoma, liposarcoma, fibrosarcoma,Ewing sarcoma and plasmocytoma. In preferred embodiments, the cancer isat least one of bone cancer, lung cancer, prostate cancer, breastcancer, ovarian cancer, and cervical cancer.

As used herein, the terms “treat”, “treatment”, and “treating” in thecontext of the administration of a therapy to a subject in need thereofrefers to the reduction or inhibition of the progression and/or durationof a disease (e.g., cancer), the reduction or amelioration of theseverity of the disease, the amelioration of one or more symptomsthereof resulting from the administration of one or more therapies,preventing the disease from occurring in a subject that may bepredisposed to the disease but does not yet experience or exhibitsymptoms of the disease (prophylactic treatment), slowing or arrestingdisease development, ameliorating the disease, providing relief from thesymptoms or side-effects of the disease (including palliativetreatment), and causing regression of the disease. Specific to cancer,and in particular bone, lung, prostate, breast, ovarian, and cervicalcancers, these terms may refer to: (1) a stabilization, reduction (e.g.,by more than 10%, 20%, 30%, 40%, 50%, preferably by more than 60% of thepopulation of cancer cells and/or tumor size before administration), orelimination of the cancer cells, (2) inhibiting cancerous cell divisionand/or cancerous cell proliferation, (3) relieving to some extent (or,preferably, eliminating) one or more symptoms associated with apathology related to or caused in part by unregulated or aberrantcellular division, (4) an increase in disease-free, relapse-free,progression-free, and/or overall survival, duration, or rate, (5) adecrease in hospitalization rate, (6) a decrease in hospitalizationlength, (7) eradication, removal, or control of primary, regional and/ormetastatic cancer, (8) a stabilization or reduction (e.g., by at least10%, 20%, 30%, 40%, 50%, 60%, 70%, preferably at least 80% relative tothe initial growth rate) in the growth of a tumor or neoplasm, (9) animpairment in the formation of a tumor, (10) a reduction in mortality,(11) an increase in the response rate, the durability of response, ornumber of patients who respond or are in remission, (12) the size of thetumor is maintained and does not increase or increases by less than 10%,preferably less than 5%, preferably less than 4%, preferably less than2%, (13) a decrease in the need for surgery (e.g., colectomy,mastectomy), and (14) preventing or reducing (e.g., by more than 10%,more than 30%, preferably by more than 60% of the population ofmetastasized cancer cells before administration) the metastasis ofcancer cells.

The term “subject” and “patient” are used interchangeably. As usedherein, they refer to any subject for whom or which therapy, includingwith the compositions according to the present disclosure is desired. Inmost embodiments, the subject is a mammal, including but not limited toa human, a non-human primate such as a chimpanzee, a domestic livestocksuch as a cattle, a horse, a swine, a pet animal such as a dog, a cat,and a rabbit, and a laboratory subject such as a rodent, e.g., a rat, amouse, and a guinea pig. In preferred embodiments, the subject is ahuman.

The subject may be any subject already with the disease, a subject whichdoes not yet experience or exhibit symptoms of the disease, or a subjectpredisposed to the disease. In preferred embodiments, the subject is aperson who is predisposed to cancer, e.g., a person with a familyhistory of cancer. Women who have (i) certain inherited genes (e.g.,mutated BRCA1 and/or mutated BRCA2), (ii) been taking estrogen alone(without progesterone) after menopause for many years (at least 5, atleast 7, or at least 10), and/or (iii) been taking fertility drugclomiphene citrate, are at a higher risk of contracting breast cancer.People who (i) consumes a diet high in salty and smoked foods and/or lowin fruits and vegetables, (ii) had infection with Helicobacter pylori,and/or (iii) long-term stomach inflammation are at a higher risk ofcontracting stomach cancer. People who (i) had chemotherapy andradiation therapy for other cancers, (ii) has genetic disorders, such asDown syndrome, and/or (iii) exposure to certain chemicals, such asbenzene are at a higher risk of contracting leukemia. People who (i) hadinflammatory bowel disease, or a genetic syndrome such as familialadenomatous polyposis (FAP) and hereditary non-polyposis colorectalcancer (Lynch syndrome), and/or (ii) consumes a low-fiber and high-fatdiet are at a higher risk of contracting colon cancer. People who havebeen diagnosed with Human papillomavirus (HPV) are at a higher risk ofcontracting cervical cancer. Any subject with such predispositions, incombination with sound medical judgment, may be candidates for thetreatment methods described herein.

In some embodiments, the subject has leukemia, stomach, colon,testicular, bladder, head and neck cancer, esophageal cancer,mesothelioma, brain, neuroblastoma, bone, lung, prostate, breast,ovarian, and/or cervical cancer and is currently undergoing, or hascompleted one or more chemotherapy regimens. In some embodiments, thesubject has been previously administered/treated with, or is beingcurrently administered/treated with, a thymidylate synthase inhibitor(e.g., capecitabine, fluorouracil (5-FU)), a thymidine phosphorylase(TPase) inhibitor (e.g., tipiracil, trifluridine), a topoisomerase Iinhibitor (e.g., irinotecan), a topoisomerase II inhibitor (e.g.,doxorubicin), a DNA synthesis inhibitor (e.g., oxaliplatin), a DNAcrosslinking agent (e.g., cisplatin), and/or a targeted therapy (e.g.,cetuximab, bevacizumab, panitumumab, zivaflibercept, ramucirumab). Insome embodiments, the subject has been previously administered/treatedwith, or is being currently administered/treated with, a tubulin bindingdrug such as paclitaxel, epothilone, docetaxel, discodermolide,etoposide, vinblastine, vincristine, teniposide, vinorelbine, andvindesine, and developed resistance to the tubulin binding drug. In someembodiments, the subject has been previously administered/treated with,or is being currently administered/treated with, a tyrosine-kinaseinhibitor such as imatinib, nilotinib, dasatinib, bosutinib, ponatinib,and bafetinib, and developed drug resistance via (i) Bcr-Abl dependentmechanisms involving Bcr-Abl duplication, Bcr-Abl mutation, T315Imutation, and/or P-loop mutations, or (ii) Bcr-Abl Independentmechanisms involving drug efflux caused by P-glycoproteins, drug importby organic cation transporter 1, and/or alternative signaling pathwayactivation. In some embodiments, the subject has been previouslyadministered/treated with, or is being currently administered/treatedwith, a DNA crosslinking agent (e.g., cisplatin) and developed drugresistance via mechanisms related to decreased intracellular uptake,increased reflux, increased inactivation by sulfhydryl molecules such asglutathione, increased excision of the adducts from DNA by repairpathways, increased lesion bypass, and/or altered expression ofregulatory proteins involved in signal transduction pathways thatcontrol the apoptotic processes. In some embodiments, the subject hasbeen previously administered/treated with, or is being currentlyadministered/treated with, a topoisomerase II inhibitor (e.g.,doxorubicin), and developed drug resistance mechanisms via alteration orincreased expression of transporters including, but not limited to, oneor more of ABCB1 (MDR1, Pgp) and ABCC1 (MRP1), as well as othertransporters.

The terms “administer”, “administering”, “administration”, and the like,as used herein, refer to the methods that may be used to enable deliveryof the active ingredient and/or the pharmaceutical composition to thedesired site of biological action. Routes or modes of administration areas set forth herein. These methods include, but are not limited to, oralroutes, intraduodenal routes, parenteral injection (includingintravenous, subcutaneous, intraperitoneal, intramuscular,intravascular, intratumoral, or infusion), topical and rectaladministration. Those of ordinary skill in the art are familiar withadministration techniques that can be employed. In preferredembodiments, the active ingredient (e.g., the gold(III) complexes) orthe pharmaceutical composition described herein are administeredparenterally, preferably as intratumoral injections, preferably as asterile isotonic aqueous or non-aqueous solution, dispersion, suspensionor emulsion.

The dosage amount and treatment duration are dependent on factors, suchas bioavailability of a drug, administration mode, toxicity of a drug,gender, age, lifestyle, body weight, the use of other drugs and dietarysupplements, the disease stage, tolerance and resistance of the body tothe administered drug, etc., and then determined and adjustedaccordingly. The terms “effective amount”, “therapeutically effectiveamount”, or “pharmaceutically effective amount” refer to that amount ofthe active ingredient being administered which will relieve to someextent one or more of the symptoms of the disease being treated. Theresult can be a reduction and/or alleviation of the signs, symptoms, orcauses of a disease, or any other desired alteration of a biologicalsystem. An appropriate “effective amount” may differ from one individualto another. An appropriate “effective amount” in any individual case maybe determined using techniques, such as a dose escalation study.Typically, an effective amount of the gold(III) complex (e.g., to treatcancers such as bone cancer, lung cancer, prostate cancer, breastcancer, ovarian cancer, and cervical cancer, in terms of mg of thegold(III) complex per body weight of the subject (kg), ranges from 0.01to 100 mg/kg, preferably 0.05 to 90 mg/kg, preferably 0.1 to 80 mg/kg,preferably 0.5 to 70 mg/kg, preferably 1 to 60 mg/kg, preferably 1.2 to50 mg/kg, preferably 1.4 to 40 mg/kg, preferably 1.6 to 30 mg/kg,preferably 1.8 to 20 mg/kg, preferably 2 to 10 mg/kg, preferably 2.2 to5 mg/kg, preferably 2.4 to 3 mg/kg, preferably 2.5 mg/kg.

Gold(III) complexes of the disclosure may be useful for sensitizingcells to apoptotic signals. Thus, in some embodiments, the gold(III)complexes of the disclosure are co-administered with radiation therapyor a second therapeutic agent with cytostatic or antineoplasticactivity. Suitable cytostatic chemotherapy compounds include, but arenot limited to (i) antimetabolites; (ii) DNA-fragmenting agents, (iii)DNA-crosslinking agents, (iv) intercalating agents (v) protein synthesisinhibitors, (vi) topoisomerase I poisons, such as camptothecinortopotecan; (vii) topoisomerase II poisons, (viii) microtubule-directedagents, (ix) kinase inhibitors (x) miscellaneous investigational agents(xi) hormones, (xii) hormone antagonists, and (xii) targeted therapies.It is contemplated that gold(III) complexes of the disclosure may beuseful in combination with any known agents falling into the above 13classes as well as any future agents that are currently in development.In particular, it is contemplated that gold(III) complexes of thedisclosure may be useful in combination with current Standards of Careas well as any that evolve over the foreseeable future. Specific dosagesand dosing regimens would be based on physicians' evolving knowledge andthe general skill in the art.

Examples of second therapeutic agents include, but are not limited to, amitotic inhibitor; an alkylating agent; an antimetabolite; a cell cycleinhibitor; an enzyme; a topoisomerase inhibitor; a biological responsemodifier; an anti-hormone; a tubulin inhibitor; a tyrosine-kinaseinhibitor; an antiangiogenic agent such as MMP-2, MMP-9 and COX-2inhibitor; an anti-androgen; a platinum coordination complex(oxaliplatin, carboplatin, cisplatin); a substituted urea such ashydroxyurea; a methylhydrazine derivative; an adrenocorticalsuppressant, e.g., mitotane, aminoglutethimide; a hormone and/or hormoneantagonist such as the adrenocorticosteriods (e.g., prednisone),progestins (e.g., hydroxyprogesterone caproate), an estrogen (e.g.,diethylstilbestrol); an antiestrogen such as tamoxifen; androgen, e.g.,testosterone propionate; and an aromatase inhibitor, such asanastrozole, and AROMASIN (exemestane); a thymidylate synthaseinhibitor; a thymidine phosphorylase (TPase) inhibitor; a DNA synthesisinhibitor; and/or a targeted therapy. Exemplary second therapeuticagents include, but are not limited to, tubulin binding agents includingpaclitaxel, epothilone, docetaxel, discodermolide, etoposide,vinblastine, vincristine, teniposide, vinorelbine, and vindesine;tyrosine-kinase inhibitors including imatinib, nilotinib, dasatinib,bosutinib, ponatinib, and bafetinib; alkylating antineoplastic agentsincluding busulfan, carmustine, chlorambucil, cyclophosphamide,cyclophosphamide, dacarbazine, ifosfamide, lomustine, mechlorethamine,melphalan, mercaptopurine, procarbazine; antimetabolites includingcladribine, cytarabine, fludarabine, gemcitabine, pentostatin,5-fluorouracil, clofarabine, capecitabine, methotrexate, thioguanine;cytotoxic antibiotics including daunorubicin, doxorubicin, idarubicin,mitomycin, actinomycin, epirubicin; topoisomerase inhibitors includingirinotecan, mitoxantrone, topotecan; thymidine phosphorylase (TPase)inhibitors such as tipiracil and trifluridine; DNA synthesis inhibitorssuch as oxaliplatin; targeted therapies such as cetuximab, bevacizumab,panitumumab, zivaflibercept, ramucirumab; and mixtures thereof.

The combination therapy is intended to embrace administration of thesetherapeutic agents in a sequential manner, that is, wherein eachtherapeutic agent is administered at a different time, as well asadministration of these therapeutic agents, or at least two of thetherapeutic agents, in a substantially simultaneous manner.Substantially simultaneous administration can be accomplished, forexample, by administering to the subject a single dosage form having afixed ratio of each therapeutic agent or in multiple, single dosageforms for each of the therapeutic agents. Sequential or substantiallysimultaneous administration of each therapeutic agent can be effected byany appropriate route including, but not limited to, oral routes,intravenous routes, intratumoral routes, intramuscular routes, anddirect absorption through mucous membrane tissues. The therapeuticagents can be administered by the same route or by different routes. Forexample, a first therapeutic agent of the combination selected may beadministered by intravenous injection while the other therapeutic agentsof the combination may be administered orally. Alternatively, forexample, all therapeutic agents may be administered orally or alltherapeutic agents may be administered by intravenous injection. Anyother administration route combination is also contemplated hereinaccording to the administration routes available for each of thetherapeutic agents. Combination therapy also can embrace theadministration of the therapeutic agents as described above in furthercombination with other biologically active ingredients and non-drugtherapies (e.g., surgery or radiation treatment). Where the combinationtherapy further comprises a non-drug treatment, the non-drug treatmentmay be conducted at any suitable time so long as a beneficial effectfrom the co-action of the combination of the therapeutic agents andnon-drug treatment is achieved. For example, in appropriate cases, thebeneficial effect is still achieved when the non-drug treatment istemporally removed from the administration of the therapeutic agents,perhaps by days or even weeks.

A treatment method may comprise administering the gold(III) complex or apharmaceutical composition containing the gold(III) complex of thecurrent disclosure in any of its embodiments as a single dose ormultiple individual divided doses. In some embodiments, the compositionis administered at various dosages (e.g., a first dose with an effectiveamount of 10 mg/kg and a second dose with an effective amount of 2mg/kg). In some embodiments, the interval of time between theadministration of the pharmaceutical composition and the administrationof one or more second therapies may be about 1 to 5 minutes, 1 to 30minutes, 30 minutes to 60 minutes, 1 hour, 1 to 2 hours, 2 to 6 hours, 2to 12 hours, 12 to 24 hours, 1 to 2 days, 2 days, 3 days, 4 days, 5days, 6 days, 7 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 15 weeks, 20 weeks, 26weeks, 52 weeks, 11 to 15 weeks, 15 to 20 weeks, 20 to 30 weeks, 30 to40 weeks, 40 to 50 weeks, 1 month, 2 months, 3 months, 4 months, 5months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12months, 1 year, 2 years, or any period of time in between. Preferably,the composition is administered once daily for at least 2 days, at least5 days, at least 6 days, or at least 7 days. In some embodiments, thepharmaceutical composition and optionally one or more second therapiesare administered less than 1 day, less than 1 week, less than 2 weeks,less than 3 weeks, less than 4 weeks, less than 1 month, less than 2months, less than 3 months, less than 6 months, less than 1 year, lessthan 2 years, or less than 5 years apart.

The methods for treating cancer and other proliferative disordersdescribed herein inhibit, remove, eradicate, reduce, regress, diminish,arrest or stabilize a cancerous tumor, including at least one of thetumor growth, tumor cell viability, tumor cell division andproliferation, tumor metabolism, blood flow to the tumor and metastasisof the tumor. In some embodiments, the size of a tumor, whether byvolume, weight or diameter, is reduced after the treatment by at least5%, at least 10%, at least 15%, at least 20%, at least 25%, at least30%, at least 40%, at least 50%, at least 60%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least99%, or 100%, relative to the tumor size before treatment. In someembodiments, the size of a tumor after treatment is not reduced but ismaintained at the same size as before treatment. Methods of assessingtumor size include, but are not limited to, CT scan, MRI, DCE-MRI, PETscan, and manual tumor measurements.

The method may further comprise measuring a concentration of a biomarkerand/or detecting a mutation in a biomarker before and/or after thepharmaceutical composition comprising the gold(III) complex of thepresent disclosure is administered. Generic cancer biomarkers includecirculating tumor DNA (ctDNA) and circulating tumor cells (CTC).Exemplary biomarkers for colon cancer include, without limitation,carcinoembryonic antigen (CEA), carbohydrate antigen 242 (CA 242), CA195, CA 19-9, MSI, and 18qLOH. Exemplary biomarkers for breast cancerinclude, without limitation, BRCA1, BRCA2, HER-2, estrogen receptor,progesterone receptor, cancer antigen 15-3, cancer antigen 27.29,carcinoembryonic antigen, Ki67, cyclin D1, cyclin E, and ERβ. Exemplarybiomarkers for stomach cancer include, without limitation,carcinoembryonic antigen (CEA), CA19-9, carbohydrate antigen (CA) 72-4,alpha-fetoprotein, carbohydrate antigen (CA)12-5, SLE, BCA-225, hCG, andpepsinogenI/II. Exemplary biomarkers for lung cancer include, withoutlimitation, CYFRA 21-1 (cytokeratins), EPCAM (epithelial cell adhesionmolecule), ProGRP (pro-gastrin-releasing peptide), and CEACAM(carcinoembryonic antigen). Exemplary biomarkers for prostate cancerinclude, without limitation, PSA, hK2/four-kallikrein panel, EN@,Annexin A3, PCA3, and TMPRSS2-ERG. Exemplary biomarkers for ovariancancer include, without limitation, CEA, cancer antigen 125 (CA125),risk of ovarian malignancy algorithm serum biomarkers (ROMA), humanepididymis protein 4 (HE4). Exemplary biomarkers for cervical cancerinclude, without limitation HPV E6, HPV E7, Mini chromosome maintenance(MCM), Cell division cycle protein 6 (CDC6), p16^(INK4A), Squamous cellcarcinoma antigen (SCC), and Ki-67.

Potentially predictive cancer biomarkers include, without limitation,mutations in genes BRCA1 and BRCA2 for breast cancer, overexpressions ofTYMS, mutations in genes p53 and KRAS for colon cancer, and highconcentration levels of AFP, and overexpressions of HSP90α for livercancer.

The mutation in the biomarker may be detected by procedures such asrestriction fragment length polymorphism (RFLP), polymerase chainreaction (PCR) assay, multiplex ligation-dependent probe amplification(MLPA), denaturing gradient gel electrophoresis (DGGE), single-strandconformation polymorphism (SSCP), hetero-duplex analysis, proteintruncation test (PTT), and oligonucleotide ligation assay (OLA). Theprocedures to detect the mutation are well-known to those of ordinaryskill in the art.

The concentration level of the cancer biomarker in a sample (i.e.,biological sample obtained from the subject in need of therapy includinga single cell, multiple cells, fragments of cells, a tissue sample,and/or body fluid, for example red blood cells, white blood cells,platelets, hepatocytes, epithelial cells, endothelial cells, a skinbiopsy, a mucosa biopsy, an aliquot of urine, saliva, whole blood,serum, plasma, lymph) may be measured for example by an immunoassay.Typical immunoassay methods include, without limitation, enzyme-linkedimmunosorbent assay (ELISA), enzyme-linked immunospot assay (ELISPOT),Western blotting, immunohistochemistry (IHC), immunocytochemistry,immunostaining, and multiple reaction monitoring (MRM) based massspectrometric immunoassay. The protocol for measuring the concentrationof the biomarker and/or detecting the mutation in the biomarker is knownto those of ordinary skill, for example by performing the steps outlinedin the commercially available assay kit sold by Sigma-Aldrich, ThermoFisher Scientific, R & D Systems, ZeptoMetrix Inc., Cayman Inc., Abcam,Trevigen, Dojindo Molecular Technologies, Biovision, and Enzo LifeSciences.

In some embodiments, a concentration of the biomarker is measured beforeand after the administration. When the concentration of the biomarker ismaintained, the method may further comprise increasing the effectiveamount of the gold(III) complex by at least 5%, at least 10%, or atleast 30%, and up to 80%, up to 60%, or up to 50% of an initialeffective amount. The subject may be administered with the increaseddosage for a longer period (e.g., 1 week more, 2 weeks more, or 2 monthsmore) than the duration prescribed with the initial effective amount.

In some embodiments, the administration is stopped once the subject istreated.

The examples below are intended to further illustrate protocols forpreparing, characterizing, and using the complexes of the presentdisclosure, and are not intended to limit the scope of the claims.

Where a numerical limit or range is stated herein, the endpoints areincluded. Also, all values and subranges within a numerical limit orrange are specifically included as if explicitly written out.

The terms “comprise(s)”, “include(s)”, “having”, “has”, “can”,“contain(s)”, and variants thereof, as used herein, are intended to beopen-ended transitional phrases, terms, or words that do not precludethe possibility of additional acts or structures. The present disclosurealso contemplates other embodiments “comprising”, “consisting of” and“consisting essentially of”, the embodiments or elements presentedherein, whether explicitly set forth or not.

As used herein, the words “a” and “an” and the like carry the meaning of“one or more.”

Obviously, numerous modifications and variations of the presentinvention are possible in light of the above teachings. It is thereforeto be understood that, within the scope of the appended claims, theinvention may be practiced otherwise than as specifically describedherein.

All patents and other references mentioned above are incorporated infull herein by this reference, the same as if set forth at length.

EXAMPLES Results

The structures of eight new gold(III) complexes (C1-C8) are shown inFIGS. 1A-1H (BPYH, 2 2′-bipyridine-3,3′-diol; BPM, 2,2′-bipyrimidine;DMDTC, dimethyldithiocarbamate; DEDTC, diethyldithiocarbamate; DBDTC,dibenzyldithiocarbamate). Four molecules (complexes C1-C4) have a2,2′-bipyridine-3,3′-diol (BPYH) moiety and a single gold atom, whilethe others (complexes C5-C8) have a 2,2′-bipyrimidine (BPM) moiety andtwo gold atoms. Complexes C2 and C6 are dimethyldithiocarbamates, C3 andC7 are diethyldithiocarbamates, and C4 and C8 aredibenzyldithiocarbamates. The gold(III) complexes had >99% purity, andinteracted with lysozyme, tryptophan and guanine (see the SupplementaryMaterials and Methods section below).

In Vitro Cytotoxicity of Gold(III) Complexes

To evaluate the potential anticancer activity of the eight complexes,their in vitro cytotoxicity was compared to that of cisplatin in a panelof cell lines derived from different human cancers including lung cancer(A549), androgen-sensitive prostate cancer (DU145), androgen-resistantprostate cancer (PC3), breast cancer (MCF-7), and osteosarcoma (MG-63)(Table 1).

TABLE 1 Half-maximal inhibitory concentrations (IC₅₀) of cisplatin andnew gold(III) complexes C1-C8 in human lung, prostate, breast andosteosarcoma cancer cell lines. Cell line Compound A549 DU145 PC3 MCF-7MG-63 Cisplatin 52.0 (4.7) 4.5 (0.4) 3.3 (0.3) 22.2 (0.2) 58.0 (0.5)C1 >80 39.0 (3.5) 28.3 (2.6) 59.0 (5.3) 43.0 (3.9) C2 6.1 (0.6) 2.8(0.3) 1.5 (0.1) 2.3 (0.2) 3.8 (0.3) C3 3.8 (0.3) 3.5 (0.3) 1.3 (0.1) 1.7(0.2) 1.2 (0.1) C4 25.0 (2.3) 6.4 (0.6) 8.5 (0.8) 13.0 (1.2) 12.3 (1.1)C5 >80 >80 >80 65.0 (5.9) 26.0 (2.3) C6 0.8 (0.1) 0.7 (0.1) 0.6 (0.1)0.5 (0.1) 0.8 (0.1) C7 1.4 (0.1) 0.8 (0.1) 0.8 (0.1) 0.6 (0.1) 0.7 (0.1)C8 23.0 (2.1) 22.8 (2.1) 19.5 (1.8) 9.5 (0.9) 5.8 (0.5) Values are mean(SD) expressed in μM.

Cisplatin had relatively low potency on three cell lines (A549, MCF-7and MG-63), with half maximal inhibitory concentrations (IC₅₀)>10 μM,while it was more potent on the DU145 and PC3 prostate cancer cell lineswith mean IC₅₀ values of 4.5 μM and 3.3 μM, respectively. Complex C1 hadIC₅₀ values higher than that of cisplatin in most cell lines, indicatinglower potency, while in MG-63 the two drugs had similar activities.Complexes C2 and C3 were more potent than cisplatin in all cell lines,and had IC₅₀ values more than one order of magnitude lower thancisplatin in the cisplatin-resistant MCF-7, A549 and MG-63 cell lines.Complexes C4 and C8 were less cytotoxic than cisplatin in PC3 and DU145cells but more cytotoxic than cisplatin in MCF-7, A549 and MG-63 cells.Complex C5 exerted very low cytotoxic effects on most cell lines exceptfor MG-63. Complex C6 and C7 had submicromolar IC₅₀ values in almost allcell lines, and thus were the most potent of all complexes tested,including cisplatin.

The cytotoxic effects of the gold(III) complexes were also evaluated intwo cell lines for which a cisplatin-resistant clone was available.First, in the ovarian cancer cell line A2780 (cisplatin sensitive),complexes C2, C3, C6 and C7 were more potent than cisplatin (i.e. theyhad IC₅₀ values<1.5 μM), whereas complexes C1, C4, C5 and C8 were lesspotent (Table 2).

TABLE 2 Half-maximal inhibitory concentrations (IC₅₀) of reference drugsand gold(III) complexes, in ovarian cancer cell line A2780 and itscisplatin- and doxorubicin-resistant A2780cis clone, and fold resistance(FR). IC50, μM^(a) FR Compound A2780 A2720cis (A2780cis/A2780) Cisplatin1.5 (0.1) 10.4 (0.9) 6.9 Doxorubicin 0.02 (0.0) 0.12 (0.0) 9.0 C1 23.0(2.1) 24.0 (2.0) 1.0 C2 0.9 (0.1) 0.8 (0.1) 0.9 C3 0.4 (0.0) 0.4 (0.0)1.1 C4 7.3 (0.7) 8.2 (0.7) 1.1 C5 15.4 (1.4) 16.2 (5.1) 1.1 C6 0.2 (0.0)0.3 (0.0) 1.2 C7 0.4 (0.0) 0.3 (0.0) 0.9 C8 3.8 (0.3) 5.2 (0.5) 1.4^(a)Mean (SD)

In the cisplatin- and doxorubicin-resistant clone A2780cis, the IC₅₀ ofeach gold(III) complex was similar to that in the parental cell line,but because the IC₅₀ of cisplatin was higher, complexes C2, C3, C4, C6,C7 and C8 were all more potent than the reference drug. The foldresistance (FR) between the two cell lines (IC₅₀ A2780cis/IC₅₀ A2780)was 6.9 for cisplatin and 9.0 for doxorubicin, while for the eight testcomplexes it was close to unity (range, 0.9 to 1.4). This resultexcludes the phenomenon of cross-resistance to these two drugs in thesecell lines.

A similar experiment was done using the ME-180 cervical cancer cell lineand its cisplatin-resistant clone R-ME-180 (Table 3). In ME-180 cells,complexes C2, C3, C6 and C7 were more potent than cisplatin (i.e. theyhad IC₅₀ values <15 μM) and complexes C1, C4, and C5 were less active.In R-ME-180 cells, the IC₅₀ value for cisplatin was higher, givingFR=4.5. The FR for the test complexes (excluding C5) was lower, rangingfrom 0.9 (C7) to 1.5 for C2.

TABLE 3 Half-maximal inhibitory concentrations (IC₅₀) of cisplatin andgold(III) complexes, in cervical cancer cell line ME-180 and itscisplatin-resistant R-ME-180 clone, and fold resistance (FR). IC₅₀,μM^(a) FR Compound ME-180 R-ME-180 (R-ME-180/ME-180) Cisplatin 15.0(1.4) 68.0 (6.1) 4.5 C1 70.0 (6.3) 72.0 (6.5) 1.0 C2 14.0 (1.3) 21.0(1.9) 1.5 C3 3.0 (0.3) 3.8 (0.3) 1.3 C4 30.0 (2.5) 30.0 (2.7) 1.0C5 >80 >80 ND C6 5.3 (0.5) 4.9 (0.5) 0.9 C7 4.8 (0.4) 4.1 (0.4) 0.9 C815.0 (1.4) 16.0 (1.4) 1.0 ND, not determined. ^(a)Mean (SD)

Then, the cellular uptake of the gold(III) complexes was examined. PC3cells were incubated separately with two low-potency complexes C4 and C5and two high-potency complexes C6 and C7, and the amount of internalizedgold was determined by mass spectrometry (FIG. 15). This analysis showedgreater uptake of the two more potent molecules, with C6 internalizationeven greater than that of C7.

To further investigate the cytotoxicity of C6 ([Au₂(BPM)(DMDTC)₂]Cl₄),its effects were compared on growth of PC3 cells and normal humanadipose-derived stromal cells. This analysis showed that C6 was morepotent in the prostate tumor cells (IC₅₀=0.6 μM) than in the normalstromal cells (IC₅₀=1.4 μM) (FIG. 16).

Altogether, these experiments show that complexes C2, C3, C6 and C7 havethe greatest potency (lowest IC₅₀ values) in the panel of investigatedtumor cell lines. Results from pairs of cell lines that differ insusceptibility to cisplatin (ME-180 and R-ME180) and also to doxorubicin(A2780 and A2780cis) rule out cross-resistance to the two chemotherapyagents.

Cellular Mechanism of Action of Complex C6

C6 was chosen for further analyses with the PC3 prostate cancer cellline. When PC3 cells were incubated with C6 at its IC₅₀ (0.62 μM) andIC₇₅ (1.85 μM), there was a dose-dependent increase in the percentage ofannexin-V-positive cells, indicating early apoptosis, and also of doublestained annexin-V- and propidium iodide (PI)-positive cells, indicatinglate apoptosis (FIGS. 2A and 2B). Consistently, treatment with C6activated caspase 3,7, evaluated using fluorochrome-labeled inhibitorsof caspases (FLICA) that irreversibly bind active caspase (FIGS. 2C and2D). These results suggest that apoptosis is involved in tumor celldeath by C6. Finally, treatment of PC3 cells with C6 modified thedistribution of cells in the cell cycle, by increasing the percentage ofcells in S phase and decreasing that in G1 compared to untreated cells(FIGS. 2E and 2F). In FIGS. 2A-2F, all bar charts report means and SD ofthree independent experiments and statistical analysis was performedusing one-way ANOVA, followed by Dunnett's multiple comparisons test,*P<0.05 vs medium.

Next, it was to be determined whether C6 treatment led PC3 cells toincrease the production of reactive oxygen species (ROS). Twoconcentrations of C6 induced ROS production in a dose-dependent manner,and this effect was blocked when cells were pretreated with N-acetylcysteine (NAC), a ROS scavenger (FIGS. 3A and 3B).

NAC decreased the cytotoxic effects of C6 (FIG. 3C), suggesting that ROSgeneration is involved in this compound's cytotoxicity. Treatment of PC3cells with C6 also induced, in a dose-dependent manner, double-strandedDNA breaks, as shown by an increase in phosphorylation of histone H2A.X(FIGS. 3D and 3E). Because ROS elimination and the maintenance ofintracellular redox balance depend on the thioredoxin (Trx) system, theeffects of C6 used at IC₂₅ (0.31 μM), IC₅₀ (0.62 μM) and IC₇₅ (1.85 μM)on Trx reductase (TrxR) levels were examined and it was found that ashort incubation resulted in a dose-dependent decrease of its enzymaticactivity (FIG. 3F). See Scalcon V.; Bindoli A.; Rigobello M. P.Significance of the mitochondrial thioredoxin reductase in cancer cells:An update on role, targets and inhibitors. Free Radic Biol Med 2018.127,62-79. Finally, C6 exerted a dose-dependent inhibitory effect alsoon 20S proteasome activity (FIG. 3G). In FIGS. 3A-3G, all bar chartsreport means and SD of three independent experiments. Statisticalanalysis was performed using one-way ANOVA, followed by Turkey's onDunnett's multiple comparisons test where appropriate. *P<0.05 vs mediumunless otherwise indicated.

Effects of C6 on Tumor Cell Migration and Xenograft Growth

The effect of C6 on PC3 cell migration was evaluated using the in vitroscratch assay. A 3 h pretreatment slowed the ability of PC3 cells torefill an empty area (“scratch”) of the monolayer-compared to untreatedcells: 36 h after the monolayer was scratched, the remaining uncoveredarea was about 40% in C6-pretreated cells, and about 10% in controlcells (FIGS. 4A and 4B). Finally, the effects of C6 on the in vivogrowth of PC3 cell xenografts in female athymic nude mice were examined.Inhibition of tumor growth became apparent starting 15 days after thebeginning of treatment, compared to animals not treated with C6 (FIG.4C). By day 32, control tumors had grown to a mean volume of 1327 mm³(SD=105 mm³) whereas C6-treated tumors reached 385 mm³ (SD=35 mm³),reflecting a 71% inhibitory effect (FIG. 4C). This difference wassignificant (P<0.0001, Student's t test). C6 treatment did not affectthe weight of the animals (FIG. 4D).

Discussion

In this study, the anticancer activity of new bipyridine andbipyrimidine gold(III) complexes (C1-C8) using a panel of cancer celllines was evaluated. The eight new complexes had potent cytotoxicity inovarian, lung, breast, prostate, cervical and sarcoma cancer cell lines.They were also active in a cisplatin-resistant cervical cell line(R-ME-180) and in a cisplatin- and doxorubicin-resistant ovarian cancercell line (A2780cis), indicating that they may overcome both cisplatinand doxorubicin resistance.

The mechanism of action and the in vivo activity of the most activecompound, C6, were evaluated using androgen-resistant PC3 prostatecancer cells. C6 induced apoptosis, activated caspases 3,7 and modifiedthe distribution of cells in cell cycle phases. Moreover, C6 increasedROS generation. ROS may play an important role in the cytotoxic effectof C6 since the ROS scavenger NAC counteracted C6's ability to inhibitcell growth. C6 treatment also induced double-stranded DNA breaks. ThisDNA damage may be due to the increased intracellular ROS levels or to apossible direct interaction of C6 with DNA. See Scalcon V.; Bindoli A.;Rigobello M. P. Significance of the mitochondrial thioredoxin reductasein cancer cells: An update on role, targets and inhibitors. Free RadicBiol Med 2018. 127,62-79, incorporated herein by reference in itsentirety.

Thioredoxin (Trx) and the seleno-enzyme thioredoxin reductase (TrxR) areessential components of the Trx system that regulates cellular redoxsignaling pathways. TrxR inhibition increases ROS accumulation, whichcauses mitochondrial dysfunction and apoptosis. See Scalcon V.; BindoliA.; Rigobello M. P. Significance of the mitochondrial thioredoxinreductase in cancer cells: An update on role, targets and inhibitors.Free Radic Biol Med 2018. 127,62-79; and Zhang J.; Li X.; Han X.; LiuR.; Fang J. Targeting the Thioredoxin System for Cancer Therapy. TrendsPharmacol Sci 2017. 38(9),794-808, each incorporated herein by referencein their entirety. High levels of Trx and TrxR have been found in manydifferent tumor types, including prostate cancer, and are associatedwith tumor progression and resistance to several anticancer drugs,including cisplatin. See Shan W.; Zhong W.; Zhao R.; Oberley T. D.Thioredoxin 1 as a subcellular biomarker of redox imbalance in humanprostate cancer progression. Free Radic Biol Med 2010. 49,2078-2087; andYamada M.; Tomida A.; Yoshikawa H.; Taketani Y.; Tsuruo T. Increasedexpression of thioredoxin/adult T-cell leukemia-derived factor incisplatin-resistant human cancer cell lines. Clin Cancer Res 1996.2,427-432, each incorporated herein by reference in their entirety. Forthese reasons, the Trx system may be a target for cancer therapy. SeeScalcon V.; Bindoli A.; Rigobello M. P. Significance of themitochondrial thioredoxin reductase in cancer cells: An update on role,targets and inhibitors. Free Radic Biol Med 2018. 127,62-79; and ZhangJ.; Li X.; Han X.; Liu R.; Fang J. Targeting the Thioredoxin System forCancer Therapy. Trends Pharmacol Sci 2017. 38(9),794-808. TrxR hasalready been identified as an important target of several gold(I) (e.g.auranofin) and gold(III) complexes. See Celegato M.; Borghese C.;Casagrande N.; Mongiat M.; Kahle X. U.; Paulitti A.; Spina M.;Colombatti A.; Aldinucci D. Preclinical activity of the repurposed drugAuranofin in classical Hodgkin lymphoma. Blood 2015. 126,1394-1397;Marzano C.; Gandin V.; Folda A.; Scutari G.; Bindoli A.; Rigobello M. P.Inhibition of thioredoxin reductase by auranofin induces apoptosis incisplatin-resistant human ovarian cancer cells. Free Radic Biol Med2007. 42(6), 872-881; Saggioro D.; Rigobello M. P.; Paloschi L.; FoldaA.; Moggach S. A.; Parsons S.; Ronconi L.; Fregona D.; Bindoli A.Gold(III)-dithiocarbamato complexes induce cancer cell death triggeredby thioredoxin redox system inhibition and activation of ERK pathway.Chem Biol 2007. 14,1128-1139; Cattaruzza L.; Fregona D.; Mongiat M.;Ronconi L.; Fassina A.; Colombatti A.; Aldinucci D. Antitumor activityof gold(III)-dithiocarbamato derivatives on prostate cancer cells andxenografts. Int J Cancer 2011. 128,206-215; and Celegato M.; Fregona D.;Mongiat M.; Ronconi L.; Borghese C.; Canzonieri V.; Casagrande N.;Nardon C.; Colombatti A.; Aldinucci D. Preclinical activity ofmultiple-target gold(III)-dithiocarbamato peptidomimetics in prostatecancer cells and xenografts. Future Med Chem 2014. 6(11),1249-1263, eachincorporated herein by reference in their entirety. Here it was foundthat, consistently with increased ROS generation, C6 inhibited TrxRenzymatic activity in PC3 cells.

The proteasome, a central component of the protein degradationmachinery, controls the expression of proteins linked to cell survivaland proliferation. See Baumann K. Protein metabolism: How the proteasomeadapts to stress. Nat Rev Mol Cell Biol 2014. 15(9),562-563,incorporated herein by reference in its entirety. Cancer cells produceanti-apoptotic and pro-survival proteins and their treatment withproteasome inhibitors causes cell cycle arrest or apoptosis, suggestingtheir use in clinic. See Manasanch E. E.; Orlowski R. Z. Proteasomeinhibitors in cancer therapy. Nat Rev Clin Oncol 2017. 14(7),417-433,incorporated herein by reference in its entirety. Some gold(III)complexes have already been found to target the proteasome in cancercells, and here it was found that C6 inhibited proteasome activity inprostate cancer cells. See Milacic V.; Chen D.; Ronconi L.;Landis-Piwowar K. R.; Fregona D.; Dou Q. P. A novel anticancer gold(III)dithiocarbamate compound inhibits the activity of a purified 20Sproteasome and 26S proteasome in human breast cancer cell cultures andxenografts. Cancer Res 2006. 66,10478-10486; Cattaruzza L.; Fregona D.;Mongiat M.; Ronconi L.; Fassina A.; Colombatti A.; Aldinucci D.Antitumor activity of gold(III)-dithiocarbamato derivatives on prostatecancer cells and xenografts. Int J Cancer 2011. 128,206-215; CelegatoM.; Fregona D.; Mongiat M.; Ronconi L.; Borghese C.; Canzonieri V.;Casagrande N.; Nardon C.; Colombatti A.; Aldinucci D. Preclinicalactivity of multiple-target gold(III)-dithiocarbamato peptidomimetics inprostate cancer cells and xenografts. Future Med Chem 2014.6(11),1249-1263; Tomasello M. F.; Nardon C.; Lanza V.; Di N. G.;Pettenuzzo N.; Salmaso S.; Milardi D.; Caliceti P.; Pappalardo G.;Fregona D. New comprehensive studies of a gold(III) Dithiocarbamatecomplex with proven anticancer properties: Aqueous dissolution withcyclodextrins, pharmacokinetics and upstream inhibition of theubiquitin-proteasome pathway. Eur J Med Chem 2017, 138,115-127; andQuero J.; Cabello S.; Fuertes T.; Marmol I.; Laplaza R.; Polo V.; GimenoM. C.; Rodriguez-Yoldi M. J.; Cerrada E. Proteasome versus ThioredoxinReductase Competition as Possible Biological Targets in Antitumor MixedThiolate-Dithiocarbamate Gold(III) Complexes. Inorg Chem 2018.57(17),10832-10845, each incorporated herein by reference in theirentirety.

Since androgen-independent prostate cancer has high invasive potential,a successful therapeutic approach should counteract not only tumorgrowth but also the metastatic potential. See Ritch C.; Cookson M.Recent trends in the management of advanced prostate cancer.F1000Research, 2018, 7 (F1000 Faculty Rev):1513, incorporated herein byreference in its entirety. Here, it was found that C6 reduced PC3 cellmigration, suggesting that this gold(III) complex may inhibit not onlytumor proliferation, but also its dissemination.

Some studies of metal-based compounds, including gold(III) complexes,have found promising in vitro cytotoxicity but did not test growthinhibition in in vivo experiments. See Nobili S.; Mini E.; Landini I.;Gabbiani C.; Casini A.; Messori L. Gold compounds as anticancer agents:chemistry, cellular pharmacology, and preclinical studies. Med Res Rev2010. 30,550-580; and Micale N.; Schirmeister T.; Ettari R.; Cinellu M.A.; Maiore L.; Serratrice M.; Gabbiani C.; Massai L.; Messori L.Selected cytotoxic gold compounds cause significant inhibition of 20Sproteasome catalytic activities. J Inorg Biochem 2014. 141,79-82, eachincorporated herein by reference in their entirety. Therefore, the invivo antitumor activity of C6 was also evaluated. Consistent with the invitro studies, C6 significantly reduced PC3 tumor xenograft growth withlow toxicity (measured as body weight change). These results arepromising for preclinical and clinical testing.

Materials and Methods

Methods for the synthesis and chemical characterization of the gold(III)complexes are described in the Supplementary Materials and Methodssection presented below, together with electrochemical methods fortesting their interactions with a protein, an amino acid, and anucleobase and cellular methods for testing uptake.

Drugs

Gold(III) complexes were dissolved in DMSO to 10 μM. The same amount ofDMSO necessary to dissolve the complexes was used as negative control inall experiments. Cisplatin and doxorubicin were surplus drugs obtainedfrom the pharmacy at Centro Riferimento Oncologico.

Cell Lines and Culture Conditions

Human androgen-resistant (PC3) and androgen-sensitive (DU145) prostatecancer cell lines were obtained from the German Collection ofMicroorganisms and Cell Cultures (DSMZ, Braunschweig, Germany). Humanbreast adenocarcinoma MCF-7 (HTB-22TM), lung cancer (A549), andosteosarcoma (MG-63) cell lines were from the American Type CultureCollection (ATCC, Rockville, USA). Human ovarian epithelialcarcinoma-derived A2780 cell line and its cisplatin- anddoxorubicin-resistant clone A2780cis were from Sigma-Aldrich. The highlyinvasive cervical cancer-derived ME-180 (HPV positive) cell line was akind gift of Dr. G. Toffoli (CRO, Aviano), and the cisplatin-resistantclone R-ME-180 was developed in our laboratory by continuous exposure to1 μM cisplatin. Cell lines were tested for mycoplasma every 15 daysusing the MycoAlert test (Lonza).

A549, MG-63, MCF-7, ME-180 and R-ME-180 cells were cultured in DMEM, andA2780, A2780cis, PC3 and DU145 cells were cultured in RPMI-1640 medium;media contained 10% heat-inactivated fetal bovine serum (FBS), 1% (v/v)of penicillin (10,000 units/mL)-streptomycin (10 mg/mL) and 1% (v/v)L-glutamine (200 mM) (all from Sigma-Aldrich). R-ME-180 and A2780ciscells were maintained in 1 μM cisplatin. Adipose-derived stromal cellswere maintained in MSCGM BulletKit medium (Lonza). All cell lines werecultured at 37° C. in a 5% CO₂, fully humidified atmosphere

Cytotoxicity Assay

Cell lines were seeded in 96-well flat-bottomed microplates in 100 μLculture medium at the following densities: DU145, PC3 and MCF-7 cells(2.5×10³ cells/well); A2780, A2780cis, ME-180, R-ME-180 and A549 cells(4.0×10³ cells/well); and MG-63 cells (2.0×10³ cells/well). Cells wereallowed to adhere for 24 h. Then the medium was replaced with freshmedium alone or with one of the gold(III) complexes at increasingconcentrations from 0 to 100 μM. The reference drugs cisplatin (0-100μM) and doxorubicin (0-1 μM) were included as positive controls forgrowth inhibition. After 72 h, cell viability was assayed using the MTTassay. All experimental conditions were tested in triplicate and theexperiment was done three times.

Half maximal inhibitory concentrations (IC₅₀, the concentration requiredfor 50% in vitro inhibition of growth) and IC₂₅ and IC₇₅ values werecalculated for each experiment using CalcuSyn software (Biosoft,Ferguson, Mo., USA). See Chou T. C.; Talalay P. Quantitative analysis ofdose-effect relationships: the combined effects of multiple drugs orenzyme inhibitors. Adv Enzyme Regul 1984. 22,27-55, incorporated hereinby reference in its entirety. IC₅₀ values were reported as mean (SD).For drug-resistance cell lines, fold resistance (FR) was calculated asthe ratio of the IC₅₀ of the resistant cell line to the IC₅₀ of theparental cell line.

Cellular Assays

In all cellular assays, PC3 cells (2.0×10⁵ cells/well in six-wellplates) were incubated in complete culture medium containing differentconcentrations of complex C6 (IC₂₅=0.31 μM, IC₅₀=0.62 μM, IC₇₅=1.85 μM).All experimental conditions were tested in triplicate and experimentswere done three times to calculate means and SD.

For apoptosis assays, PC3 cells were treated with C6 for 24 h and thenapoptosis was assayed by staining for 15 min with FITC Annexin V reagent(BD Pharmingen) and propidium iodide (PI). Apoptotic cells were detectedby flow cytometry (BD FACSCanto II flow cytometer) and analyzed using BDFACSDiva v8.0.1 software (BD Biosciences). Caspase 3,7 activation wasevaluated using fluorochrome-labeled inhibitors of caspases (FLICA) ofthe CaspaTag Caspase 3,7 In Situ Assay Kit, Fluorescein (Millipore) andevaluated by flow cytometry; data were expressed as mean fluorescenceintensity.

To assay the distribution of cells in the various phases of the cellcycle, PC3 cells were treated with C6 for 48 h, then harvested, fixed incold 70% ethanol for 15 min and stained with PI solution (50 μg/mL PI,0.1% NP-40, 100 μg/mL PureLink RNase A, 0.1% sodium citrate). After 1 h,cells were analyzed by flow cytometry. The distribution of cells indifferent cell cycle phases was quantified using ModFit LT 4.0 software.

The production of reactive oxygen species (ROS) was evaluated using2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) (H2-DCF, DCF)(Invitrogen). Cells were pretreated with the antioxidantN-acetyl-L-cysteine (NAC; 5 mM) (Sigma) for 30 min before C6 was added.After 24 h of C6 treatment, cells were harvested and viable cells werecounted by trypan blue dye exclusion. Then cells were washed, stainedwith 1 μM H2DCFDA for 30 min at 37° C., and finally ROS production wasanalyzed by flow cytometry.

The presence of double-stranded DNA breaks was assessed 24 h aftertreatment with C6 by fixing and permeabilizing cells with Fix & Permmedium A and B (Invitrogen) and staining with FITC anti-H2A.X Phospho(Ser139) Antibody (BioLegend, San Diego, USA). Stained cells wereevaluated by flow cytometry.

Thioredoxin reductase (TrxR) (EC 1.8.1.9) was assayed using theThioredoxin Reductase Assay Kit (Sigma-Aldrich). Cells were treated withC6 for 12 h and then lysed in 50 mM Tris-HCl pH 7.6, 0.1% Triton X-100,0.9% NaCl. Enzyme activity was determined reading absorbance at 412 nmusing a spectrophotometer (Biomate 3 Thermo Spectronic). The enzymaticactivity was normalized to the protein concentration, determined usingthe Bio-Rad protein assay (Protein Assay Dye Reagent Concentrate,Bio-Rad Laboratories), and expressed as percentage of control (no C6).

Proteasome activity (EC 3.4.25.1) was evaluated on the same cell lysatesas used to assay TrxR. Proteasome activity was assayed in cytosolicextracts using the 20S Proteasome Activity Assay kit APT280 (MerckMillipore) and a computer-interfaced GeniusPlus microplate reader(Tecan). The activity was normalized to the protein concentration,determined using the Bio-Rad protein assay, as expressed as percentageof control.

Cell migration was assessed using the in vitro scratch assay. Briefly,cells were grown to confluence and then treated with C6 (IC₅₀). After 3h, monolayers were washed twice with PBS, scraped with a pipette tip tocreate a “wound” in the monolayer, and washed again. Culture medium with2% (not 10%) FBS was added and the cells were cultured for 36 h. Woundswere photographed every 12 h using an inverted microscope(EclipseTS/100, Nikon) at magnification 4×. Migration was assessed bymeasuring the cell-free area (in pixels) with ImageJ tool software after12, 24 and 36 h.

Human Prostate Tumor Xenograft Experiments

Animal experiments were approved by the Italian Ministry of Health (no.671/2015/PR). Ten 4-week-old female athymic nu/nu (nude) mice werepurchased from Envigo. PC3 cells (3×10⁶ in a 0.1 mL solution of Matrigel1:3 in PBS) were inoculated subcutaneously into the right flank of eachmouse. Body weight and tumors were measured three times a week, andtumor volumes were calculated according to the formula:(width²×length×3.14)/6. When tumors reached a volume of ca. 120 mm³,mice were divided into two groups of five animals each. Mice weretreated every other day with an intratumoral injection of 2.5 mg/kg C6or an equal volume of vehicle (10% DMSO, 20% Cremophor Sigma-Aldrich,70% PBS). Mice were killed on day 32 when control tumors had reachedabout 1300 mm³.

Statistical Analysis

Statistical analysis was performed using GraphPad Prism v6 software.Student's t test was used to compare two groups, and one-way analysis ofvariance (ANOVA) was used for three or more groups; consecutive multiplecomparisons were performed using Dunnett's or Tukey's test. P<0.05indicated statistical significance.

Supplementary Materials and Methods

Materials and methods used in the synthesis and chemicalcharacterization of gold(III) complexes (C1-C8), and electrochemicalmethods and methods for cellular uptake assays are presented below.Additionally, results are presented on the yield and purity of gold(III)complexes, electrochemical data on the interactions of gold(III)complexes with lysozyme (Table 4 and FIGS. 5A-5D, FIGS. 6A-6D, FIGS.7A-7D, FIGS. 8A-8D), tryptophan (Table 5 and FIGS. 9A-9D, FIGS. 10A-10D,FIGS. 11A-11D, FIGS. 12A-12D) and guanine (Table 6 and FIGS. 13A-13F andFIGS. 14A-14F), and cellular data on the uptake of selected gold(III)complexes by PC3 cells (FIG. 15) and on the inhibition of growth by C6in PC3 prostate cancer cells and adipose-derived stromal cells (FIG.16).

Reagents

Sodium tetrachloroaurate(III) dihydrate, sodium dimethyldithiocarbamatehydrate, sodium diethyldithiocarbamate trihydrate, sodiumdibenzyldithiocarbamate hydrate, 2,2′-bipyrimidine,2,2′-bipyridine-3,3′-diol, disodium hydrogen phosphate, sodiumdihydrogen phosphate, tryptophan, lysozyme, 98% guanine, and 99.8%ethanol were obtained from Sigma-Aldrich. Anhydrous 99.8%dichloromethane were purchased from Merck and used without furtherpurification. Double distilled water used only for electrochemicalmeasurements was from an Aquatron A4000D water still (Stuart). Allreactions were carried out at ambient room temperature.

Synthesis of Gold(III) Complexes (C1-C8)

[Au(BPYH)(Cl)₂]Cl (C1) was synthesized by combining 0.5 mMNa[AuCl₄].2H₂O (200 mg in 3 mL H₂O) and 0.5 mM 2 2′-bipyridine-3 3′-diol(94 mg in 15 mL of ethanol:dichloromethane (3:1)) and stirred for 3 h.The solution was filtered and kept in an undisturbed area for 3 days.Black cubic crystals appeared. The black precipitate was collected byfiltration, washed with distilled water (3×10 mL) and dried undervacuum.

[Au(BPYH)(DMDTC)]Cl₂ (C2) was synthesized stepwise. First, 0.5 mM

Na[AuCl₄]·2H₂O (200 mg in 3 mL distilled water) and 0.5 mM 22′-bipyridine-3 3′-diol (94.0 mg in 15 mL ethanol:dichloromethane (3:1))were added simultaneously to 20 mL of 99.8% ethanol, and the mixture wasstirred for 3 h, generating a pale yellow, turbid solution. Second, 0.5mM sodium dimethyldithiocarbamate hydrate (71.6 mg in 10 mL distilledwater) was added dropwise and the mixture was stirred for an additional1 h. The product light-yellow precipitate was collected by filtration,washed with distilled water (3×10 mL), and dried under vacuum.

[Au(BPYH)(DEDTC)]Cl₂ (C3) was synthesized stepwise. First, 0.5 mMNa[AuCl₄]·2H₂O (200 mg in 3 mL H₂O) and 0.5 mM 2 2′-bipyridine-3 3′-diol(94.0 mg in 15 mL ethanol: dichloromethane (3:1)) were addedsimultaneously to 20 mL of 99.8% ethanol, and the mixture was stirredfor 3 h, generating a yellow turbid solution. Second, 0.5 mM sodiumdiethyldithiocarbamate trihydrate (112.6 mg in 10 mL distilled water)was added dropwise and the mixture was stirred for an additional 1 h.The obtained yellow precipitate was collected by filtration, washed withdistilled water (3×10 mL) and dried under vacuum. The final product wasa yellow crystalline powder.

[Au(BPYH)(DBDTC)]Cl₂ (C4) was synthesized stepwise. First, 0.5 mM 22′-bipyridine-3 3′-diol (94.0 mg in 15 mL ethanol:dichloromethane (3:1))was combined with 0.5 mM Na[AuCl₄]·2H₂O (200 mg in 3 mL distilled water)and the mixture was stirred for 3 h, generating an orange turbidsolution. Second, 0.5 mM sodium dibenzyldithiocarbamate hydrate (148 mgin 10 mL 99.8% ethanol) was added dropwise and the mixture was stirredfor an additional 1 h. The orange precipitate was collected byfiltration, washed with distilled water (3×10 mL) and dried undervacuum. The final product was an orange crystalline powder.

[Au₂(BPM)(Cl)₄]Cl₂ (C5) was synthesized by combining 0.5 mM 22′-bipyrimidine (79 mg in 20 mL 99.8% ethanol) and 1.0 mM Na[AuCl₄]·2H₂O(397.8 mg in 10 mL distilled water). The mixture was stirred for 3 h.The yellow precipitate was collected by filtration, washed withdistilled water (3×10 mL), and dried under vacuum.

[Au₂(BPM)(DMDTC)₂]Cl₄ (C6) was synthesized stepwise. First, 0.5 mM 22′-bipyrimidine (79 mg in 20 mL 99.8% ethanol) was combined with 1.0 mMNa[AuCl₄]·2H₂O (397.8 mg in 3 mL distilled water). The mixture wasstirred for 3 h, generating a bright yellow, turbid solution. Then, 1.0mM sodium dimethyldithiocarbamate hydrate (143.2 mg in 20 mL distilledwater) was slowly added, and the reaction mixture was stirred for 1 h.The product appeared as a pale yellow precipitate. The precipitate wascollected by filtration, washed with distilled water (3×10 mL) and driedunder vacuum.

[Au₂(BPM)(DEDTC)₂]Cl₄ (C7) was synthesized stepwise. First, 0.5 mM2,2′-bipyrimidine (79 mg in 20 mL 99.8% ethanol) was combined with 1.0mM Na[AuCl₄]2H₂O (397.8 mg in 3.0 mL distilled water). The mixture wasstirred for 3 h, generating a bright yellow, turbid solution. Then, 1.0mM sodium diethyldithiocarbamate hydrate (226 mg in 20 mL distilledwater) was slowly added, and the reaction mixture was stirred for 1 h.The product appeared as a dark yellow precipitate; it was collected byfiltration, washed with distilled water (3×10 mL), and dried undervacuum for 72 hours.

[Au₂(BPM)(DBDTC)₂]Cl₄ (C8) was synthesized stepwise. First, 0.5 mM 22′-bipyrimidine (79 mg in 20 mL 99.8% ethanol) was combined with 1.0 mMNa[AuCl₄]·2H₂O (397.8 mg in 3 mL distilled water). The mixture wasstirred for 3 h, generating a yellow, turbid solution. Then, 1.0 mMsodium dibenzyldithiocarbamate hydrate (295.4 mg in 20 mL distilledwater) was slowly added, and the reaction mixture was stirred for 1 h.The product appeared as a yellowish green precipitate; it was collectedby filtration, washed with distilled water (3×10 mL), and dried undervacuum for 72 hours.

Chemical Characterization of Gold(III) Complexes

Due to the poor solubility of the gold(III) complexes in water, theywere dissolved in 99.8% ethanol. The pH of buffers was monitored on aAccumet XL50 pH meter. A GR-2000 electrical balance was used to weighthe various chemicals. Electrochemical measurements for cyclicvoltammetry and square wave voltammetry were performed using Autolabinstruments (Metrohm; Netherlands). The electrochemical workstation hadthree electrodes (from CH Instruments): a glassy carbon electrode (GCE)as the working electrode, platinum as the counter electrode, and Ag/AgClas the reference electrode (in saturated KCl). The GCE was polished as amirror-like surface with alumina slurry on a synthetic cloth beforeevery electrochemical analysis. Square wave voltammetry and cyclicvoltammetry were scanned from 0 to 1.3 V for the various analyses.Elemental analyses of gold(III) complexes (C1-C8) were performed onPerkinElmer Series 11 (CHNS/O), Analyzer 2400.

The solid state FTIR spectra of sodium dimethyldithiocarbamate hydrate,sodium diethyldithiocarbamate trihydrate, and sodiumdibenzyldithiocarbamate hydrate (free ligands) and their correspondinggold(III) complexes were recorded on a PerkinElmer FTIR 180spectrophotometer or NICOLET 6700 FTIR using potassium bromide (KBr)pellets over the range 4000-400 cm⁻¹. ¹H and ¹³C NMR spectra wererecorded on a LAMBDA 500 spectrophotometer operating at 500.01 and125.65 MHz respectively, corresponding to a magnetic field of 11.74 T.Tetramethylsilane was used as an internal standard for ¹H and ¹³C. The¹³C NMR spectra were obtained with ¹H broadband decoupling, and thespectral conditions were: 32 k data points, 0.967 s acquisition time,1.00 s pulse delay and 45 g° pulse angle.

Gold(III) Complex Interactions with Lysozyme, Tryptophan and Guanine

The electrochemical investigation of the interactions between thegold(III) complexes (C1-C8) and lysozyme, tryptophan and guanine wasperformed using the Autolab instrument described above, with athree-electrode system (CH Instruments): platinum wire counter electrode(CHI115), Ag/AgCl reference electrode (in 3 M KCl, CHI111) and glassycarbon working electrode (CHI112) inserted into a 5.0 ml glass cell.Solutions of 1 mM lysozyme, 5 mM tryptophan and 5 mM guanine wereprepared in double distilled water, and the experiment was performed in0.1 M phosphate buffer at pH 6.8.

Cellular Uptake of Gold(III) Complexes

PC3 cells (1×10⁶ cells seeded in 100×20 culture dishes) were treated for2 h in duplicate with 3 μM C4, C5, C6 or C7 in complete culture medium.After treatment, monolayers were washed with ice-cold PBS four times,and the cells were detached with trypsin-EDTA and washed three timeswith ice-cold PBS by centrifugation. The cell pellet was solubilized in700 μL of HNO₃—HCl solution (1:3 molar ratio) for 2 h at 100° C., andthen diluted with 4 mL water. Samples were analyzed for gold on anAgilent 7500 inductively coupled plasma mass spectrometer (ICP-MS).Results were expressed as ng gold/10⁶ cells. The experiment wasperformed a total of two times and the results were expressed as meanand SD.

Growth Inhibition Curves for C6 in Adipose-Derived Stromal Cells

Human adipose-derived stromal cells (ADSCs) were from Lonza (Verviers,Belgium). ADSCs were maintained in MSGM bullet kit (Lonza) andexperiments were performed in DMEM (Cambrex Bio Science, Milan, Italy)supplemented with 10% FBS. To evaluate effects of C6, ADSCs were seededin 96-well flat-bottomed microplates (5.0×10³ cells in 100 μL per well)and incubated for 24 h (to allow cell adhesion) before drug testing. Themedium was removed and replaced with fresh medium containing C6 atincreasing concentrations (from 0.1 to 1 μM). Cells were incubated at37° C. for 72 h. Each treatment was performed in triplicate. Cell growthwas measured using the MTT assay.

Supplementary Results

Analytical Data of Gold(III) Complexes

[Au(BPYH)(Cl)₂]Cl (C1). Yield: 83.09% (219.98 mg). FT-IR (KBr, υ_(max),cm⁻¹): 3470 (b), 3071 (w), 1647 (m), 1584 (s), 1459 (s), 1269 (m), 1134(s), 1012 (m), 916 (w), 790 (s), 565 (m), 508 (m). ¹H NMR (500 MHz,DMSO-d₆): δ=7.57, 8.25 and 8.77 (3H, 2×CH, BPYH). ¹³C NMR (125.65 MHz,DMSO-d₆): δ=125.81, 128.31, 136.68, 138.63 and 155.31 (BPYH). Anal.calc. for C₁₀H₇Cl₂N₂O₂Au (456); C, 26.38; H, 1.55; N, 6.16; Found: C,26.42; H, 1.53; N, 6.18%.

[Au(BPYH)(DMDTC)]Cl₂ (C2). Yield: 83.09% (219.98 mg). FT-IR (KBr,υ_(max), cm⁻¹): 3437 (b), 3055 (w), 2921 (w); 1576 (s), 1484 (s), 1299(m), 1111 (w), 1060 (m), 989 (w), 795 (s), 580 (m). ¹H NMR (500 MHz,DMSO-d₆): δ=2.49 (6H, 2×CH₃), 7.51, 8.18 and 8.63 (2H, 2×CH, BPYH). ¹³CNMR (125.65 MHz, DMSO-d₆): δ=39.38 (CH₃), 126.20, 128.30, 135.49, 138.74and 155.33 (2,2′-BPYH), 193.84 (NC═S). Anal. calc. for C₁₃H₁₃Cl₁N₃O₂S₂Au(539.81): C, 28.92; H, 2.43; N, 7.79; S, 11.88%. Found: C, 28.97; H,2.39; N, 7.81; S, 11.91%.

[Au(BPYH)(DEDTC)]Cl₂ (C3). Yield: 86.87% (166.87 mg). FT-IR (KBr,υ_(max), cm⁻¹): 3443 (b), 3047 (w), 2928 (w), 1572 (s), 1490 (s), 1235(m), 1155 (w), 1063 (m), 876 (m), 796 (s), 548 (m). ¹H NMR (500 MHz,DMSO-d₆): δ=2.49 (6H, 2×CH₃), 3.75 (4H, 2×CH₂), 7.49, 8.18 and 8.62 (2H,2×CH, BPYH). ¹³C NMR (125.65 MHz, DMSO-d₆): δ=12.11 (CH₃), 46.52 (CH₂),125.78, 128.21, 136.74, 138.79 and 155.33 (2,2′-BPYH), 195.11 (NC═S).Anal. calc. for C₁₅H₁₇Cl₁N₃O₂S₂Au (567.86): C, 31.73; H, 3.02; N, 7.40;S, 11.29%. Found: C, 31.76; H, 2.99; N, 7.43; S, 11.31%.

[Au(BPYH)(DBDTC)]Cl₂ (C4). Yield: 85.55% (226.13 mg). FT-IR (KBr,υ_(max), cm⁻¹): 3444 (b), 3021 (w), 2925 (w); 1532 (s), 1434 (s), 1223(s), 1117 (m), 1068 (m), 982 (m), 796 (s), 549 (m). ¹H NMR (500 MHz,DMSO-d₆): δ=5.78 (4H, 2×CH₂), 8.10 (10H, 2×C₆H₅), 8.18, 8.27 and 8.93(2H, 2×CH, BPYH). ¹³C NMR (125.65 MHz, DMSO-d₆): δ=55.01 (CH₂), 125.67,128.93, 138.78 and 155.35 (BPYH), 128.21-132.49 (C₆H₅), 199.10 (NC═S).Anal. calc. for C₂₅H₂₁Cl₁N₃O₂S₂Au (692.00): C, 43.39; H, 3.06; N, 6.07;S, 9.27%. Found: C, 43.41; H, 3.05; N, 6.09; S, 9.31%.

[Au₂(BPM)(Cl)₄]Cl₂ (C5). Yield: 80.09% (187.27 mg). FT-IR (KBr, υ_(max),cm⁻¹): 3073 (m), 1577 (s), 1406 (s), 1226 (m), 1113 (m), 1023 (m), 821(m), 570 (m). ¹H NMR (500 MHz, DMSO-d₆): δ=7.35 and 8.72 (4H, 4×CH and2H, 2×CH, BPM). ¹³C NMR (125.65 MHz, DMSO-d₆): δ=121.99, 157.89 and161.84 (BPM). Anal. calc. for C₈H₆Cl₆N₄Au₂ (764.81): C, 12.56; H, 0.79;N, 7.33%. Found: C, 12.59; H, 0.78; N, 7.35%.

[Au₂(BPM)(DMDTC)₂]Cl₄ (C6). Yield: 80.09% (187.27 mg). FT-IR (KBr,υ_(max), cm⁻¹): 3057 (w), 2925 (w), 1578 (s), 1402 (s), 1238 (m), 1163(m), 1046 (m), 967 (w), 876 (w), 559 (m). ¹H NMR (500 MHz, DMSO-d₆):δ=2.35 (6H, 2×CH₃), 7.35 and 8.70 (4H, 4×CH and 2H, 2×CH, BPM). ¹³C NMR(125.65 MHz, DMSO-d₆): δ=40.29 (CH₃), 121.28, 135.33, 140.27 and 147.80(2,2′-BPM), 193.87 (NC═S). Anal. calc. for C₁₄H₁₈Cl₄N₆S₄Au₂ (934.34): C,18.00; H, 1.94; N, 8.99; S, 13.73%. Found: C, 18.05; H, 1.91; N, 9.03;S, 13.77%.

[Au₂(BPM)(DEDTC)₂]Cl₄ (C7). Yield: 88.51% (343.9 mg). FT-IR (KBr,υ_(max), cm⁻¹): 3055 (w), 2978 (w), 2930 (w), 1552 (s), 1463 (s), 1351(m), 1286 (s), 1195 (m), 1089 (m), 994 (m), 846 (m), 584 (m). ¹H NMR(500 MHz, DMSO-d₆): δ=2.43 (6H, 2×CH₃), 3.85 (4H, 2×CH₂), δ=7.33 and8.71 (4H, 4×CH and 2H, 2×CH, BPM). ¹³C NMR (125.65 MHz, DMSO-d₆):δ=12.13 (CH₃), 46.58 (CH₂), 120.55, 156.63 and 161.75 (BPM), 193.89(NC═S). Anal. calc. for C₁₈H₂₆Cl₄N₆S₄Au₂ (990.44): C, 21.83; H, 2.65; N,8.49; S, 12.95%. Found: C, 21.88; H, 2.61; N, 8.55; S, 13.07%.

[Au₂(BPM)(DBDTC)₂]Cl₄ (C8). Yield: 78.01% (256.6 mg). FT-IR (KBr,υ_(max), cm⁻¹): 3051 (w), 2972 (w), 2922 (m), 1573 (s), 1471 (s), 1355(m), 1234 (s), 1133 (m), 1047 (m), 980 (s), 553 (m). ¹H NMR (500 MHz,DMSO-d₆): δ=5.02 (4H, 2×CH₂), 7.37 (10H, 2×C₆H₅), 7.33 and 8.75 (4H,4×CH and 2H, 2×CH, BPM). ¹³C NMR (125.65 MHz, DMSO-d₆): δ=55.37 (CH₂),120.81, 156.23 and 161.90 (BPM), 128.17-132.50 (C₆H₅), 199.14 (NC═S).Anal. calc. for C₃₈H₃₄Cl₄N₆S₄Au₂ (1238.72): C, 36.85; H, 2.77; N, 6.78;S, 10.35%. Found: C, 36.88; H, 2.73; N, 6.80; S, 10.37%.

FIG. 5A shows cyclic voltammetry of 100 μM C1 with varyingconcentrations of lysozyme: (a) buffer blank; (b) C1 alone; (c) C1 and 1μM lysozyme; (d) C1 and 4 μM lysozyme; and (e) C1 and 10 μM lysozyme,FIG. 5B shows square-wave voltammetry of 100 μM C1 with variousconcentrations of lysozyme as in FIG. 5A, FIG. 5C shows cyclicvoltammetry of 100 μM C1 in control experiments with varying volumes ofdouble-distilled water: (a) buffer blank; (b) 0 μL; (c) 3 μL; (d) 12 μL;and (e) 30 μL, and FIG. 5D shows square-wave voltammetry of 100 μM C1 incontrol experiments with varying volumes of double-distilled water as inFIG. 5C.

FIG. 6A shows cyclic voltammetry of 100 μM C2 with varyingconcentrations of lysozyme: (a) buffer blank; (b) C2 alone; (c) C2 and 1μM lysozyme; (d) C2 and 4 μM lysozyme; and (e) C2 and 10 μM lysozyme,FIG. 6B shows square-wave voltammetry of 100 μM C2 with variousconcentrations of lysozyme as in FIG. 6A, FIG. 6C shows cyclicvoltammetry of 100 μM C2 in control experiments with varying volumes ofdouble-distilled water: (a) buffer blank; (b) 0 μL; (c) 3 μL; (d) 12 μL;and (e) 30 μL, and FIG. 6D shows square-wave voltammetry of 100 μM C2 incontrol experiments with varying volumes of double-distilled water as inFIG. 6C.

FIG. 7A shows cyclic voltammetry of 100 μM C3 with varyingconcentrations of lysozyme: (a) buffer blank; (b) C3 alone; (c) C3 and 1μM lysozyme; (d) C3 and 4 μM lysozyme; and (e) C3 and 10 μM lysozyme,FIG. 7B shows square-wave voltammetry of 100 μM C3 with variousconcentrations of lysozyme as in FIG. 7A, FIG. 7C shows cyclicvoltammetry of 100 μM C3 in control experiments with varying volumes ofdouble-distilled water: (a) buffer blank; (b) 0 μL; (c) 3 μL; (d) 12 μL;and (e) 30 μL, and FIG. 7D shows square-wave voltammetry of 100 μM C3 incontrol experiments with varying volumes of double-distilled water as inFIG. 7C.

FIG. 8A shows cyclic voltammetry of 100 μM C4 with varyingconcentrations of lysozyme: (a) buffer blank; (b) C4 alone; (c) C4 and 1μM lysozyme; (d) C4 and 4 μM lysozyme; and (e) C4 and 10 μM lysozyme,FIG. 8B shows square-wave voltammetry of 100 μM C4 with variousconcentrations of lysozyme as in FIG. 8A, FIG. 8C shows cyclicvoltammetry of 100 μM C4 in control experiments with varying volumes ofdouble-distilled water: (a) buffer blank; (b) 0 μL; (c) 3 μL; (d) 12 μL;and (e) 30 μL and FIG. 8D shows square-wave voltammetry of 100 μM C4 incontrol experiments with varying volumes of double-distilled water as inFIG. 8C.

FIG. 9A shows cyclic voltammetry of 0.5 mM tryptophan with varyingconcentrations of C5: (a) buffer blank; (b) tryptophan alone; (c)tryptophan and 10 μM C5; (d) tryptophan and 40 μM C5; and (e) tryptophanand 100 μM C5, FIG. 9B shows square-wave voltammetry of 0.5 mMtryptophan with varying concentrations of C5 as in FIG. 9A, FIG. 9Cshows cyclic voltammetry of 0.5 mM tryptophan in control experimentswith varying volumes of ethanol: (a) buffer blank; (b) 0 μL; (c) 15 μL;(d) 60 μL; and (e) 150 μL, and FIG. 9D shows square-wave voltammetry of100 μM C5 in control experiments with varying volumes of ethanol as inFIG. 9C.

FIG. 10A shows cyclic voltammetry of 0.5 mM tryptophan with varyingconcentrations of C6: (a) buffer blank; (b) tryptophan alone; (c)tryptophan and 10 μM C6; (d) tryptophan and 40 μM C6; and (e) tryptophanand 100 μM C6. FIG. 10B shows square-wave voltammetry of 0.5 mMtryptophan with varying concentrations of C6 as in FIG. 10A. FIG. 10Cshows cyclic voltammetry of 0.5 mM tryptophan in control experimentswith varying volumes of ethanol: (a) buffer blank; (b) 0 μL; (c) 15 μL;(d) 60 μL; and (e) 150 μL, and FIG. 10D shows square-wave voltammetry of100 μM C6 in control experiments with varying volumes of ethanol as inFIG. 10C.

FIG. 11A shows cyclic voltammetry of 0.5 mM tryptophan with varyingconcentrations of C7: (a) buffer blank; (b) tryptophan alone; (c)tryptophan and 10 μM C7; (d) tryptophan and 40 μM C7; and (e) tryptophanand 100 μM C7, FIG. 11B shows square-wave voltammetry of 0.5 mMtryptophan with varying concentrations of C7 as in FIG. 11A, FIG. 11Cshows cyclic voltammetry of 0.5 mM tryptophan in control experimentswith varying volumes of ethanol: (a) buffer blank; (b) 0 μL; (c) 15 μL;(d) 60 μL; and (e) 150 μL and FIG. 11D shows square-wave voltammetry of100 μM C7 in control experiments with varying volumes of ethanol as inFIG. 11C.

FIG. 12A shows cyclic voltammetry of 0.5 mM tryptophan with varyingconcentrations of C8: (a) buffer blank; (b) tryptophan alone; (c)tryptophan and 10 μM C8; (d) tryptophan and 40 μM C8; and (e) tryptophanand 100 μM C8, FIG. 12B shows square-wave voltammetry of 0.5 mMtryptophan with varying concentrations of C8 as in FIG. 12A, FIG. 12Cshows cyclic voltammetry of 0.5 mM tryptophan in control experimentswith varying volumes of ethanol: (a) buffer blank; (b) 0 μL; (c) 15 μL;(d) 60 μL; and (e) 150 μL, and FIG. 12D shows square-wave voltammetry of100 μM C5 in control experiments with varying volumes of ethanol as inFIG. 12C.

FIG. 13A shows cyclic voltammetry of 0.5 mM guanine with varyingconcentrations of C1: (a) buffer blank; (b) guanine alone; (c) guanineand 10 μM Cl; (d) guanine and 40 μM C1; and (e) guanine and 100 μM C1,FIG. 13B shows square-wave voltammetry of 0.5 mM guanine with varyingconcentrations of C1 as in FIG. 13A, FIG. 13C shows cyclic voltammetryof 0.5 mM guanine in control experiments with varying volumes ofethanol: (a) buffer blank; (b) 0 μL; (c) 15 μL; (d) 60 μL; and (e) 150μL, FIG. 13D shows square-wave voltammetry of 0.5 mM guanine in controlexperiments with varying volumes of ethanol: (a) buffer blank; (b) 0 μL;(c) 15 μL; (d) 60 μL; and (e) 150 μL, FIG. 13E shows cyclic voltammetryof C1 alone at: (a) 0 μM; (b) 10 μM; (c) 40 μM; and (d) 100 μM, and FIG.13F shows square-wave voltammetry of C1 alone at: (a) 0 μM; (b) 10 μM;(c) 40 μM; and (d) 100 μM.

FIG. 14A shows cyclic voltammetry of 0.5 mM guanine with varyingconcentrations of C5: (a) buffer blank; (b) guanine alone; (c) guanineand 10 μM C5; (d) guanine and 40 μM C5; and (e) guanine and 100 μM C5,FIG. 14B shows square-wave voltammetry of 0.5 mM guanine with varyingconcentrations of C5 as in FIG. 14A, FIG. 14C shows cyclic voltammetryof 0.5 mM guanine in control experiments with varying volumes ofethanol: (a) buffer blank; (b) 0 μL; (c) 15 μL; (d) 60 μL; and (e) 150μL, FIG. 14D Square-wave voltammetry of 0.5 mM guanine in controlexperiments with varying volumes of ethanol: (a) buffer blank; (b) 0 μL;(c) 15 μL; (d) 60 μL; and (e) 150 μL, FIG. 14E shows cyclic voltammetryof C5 alone at: (a) 0 μM; (b) 10 μM; (c) 40 μM; and (d) 100 μM, and FIG.14F shows square-wave voltammetry of C5 alone at: (a) 0 μM; (b) 10 μM;(c) 40 μM; and (d) 100 μM.

TABLE 4 Voltammetry peak potential (mV) of 100 μM complexes (C1-C4) with1-10 μM lysozyme Lysozyme Compound Technique 0 μM 1 μM 4 μM 10 μM C1 CV0.73898 0.81421 0.83191 0.87616 C1 SWV 0.79338 0.80345 0.8387  0.86388C2 CV 0.79208 0.80093 0.81863 0.83191 C2 SWV 0.78835 0.79338 0.808490.81352 C3 CV 0.79651 0.80978 0.82306 0.83633 C3 SWV 0.79338 0.803450.81352 0.82359 C4 CV 0.79651 0.80978 0.81863 0.82748 C4 SWV 0.783350.79338 0.80849 0.81352 CV, cyclic voltammetry; SWV, square-wavevoltammetry

TABLE 5 Voltammetry peak potential (mV) of 0.5 mM tryptophan with 10-100μM complexes (C5-C8) Compound Compound Technique 0 μM 1 μM 40 μM 10 μMC5 CV 0.71243 0.72571 0.74341 0.74783 C5 SWV 0.70862 0.71365 0.738830.7489  C6 CV 0.71243 0.73013 0.73898 0.74341 C6 SWV 0.70862 0.718690.72372 0.72876 C7 CV 0.71243 0.72571 0.75668 0.78323 C7 SWV 0.708620.71365 0.72876 0.7338  C8 CV 0.71243 0.71686 0.72128 0.78323 C8 SWV0.70862 0.70862 0.71365 0.71869 CV, cyclic voltammetry; SWV, square-wavevoltammetry

TABLE 6 Voltammetry peak potential (mV) of 0.5 mM guanine with 10-100 μMcomplexes C1 and C5 Compound Compound Technique 0 μM 10 μM 40 μM 100 μMC1 CV 0.93811 0.96024 0.98236 1.0222 C1 SWV 0.93437 0.94444 0.959550.9948 C5 CV 0.93811 0.99564 1.0133  1.0753 C5 SWV 0.93437 0.9948 1.025   1.0502 CV, cyclic voltammetry; SWV, square-wave voltammetry

1. A gold(III) complex of formula (I),

or a pharmaceutically acceptable salt, solvate, tautomer, orstereoisomer thereof, wherein: R¹ and R² are each independently ahydrogen, an optionally substituted alkyl, an optionally substitutedcycloalkyl, an optionally substituted arylalkyl, or an optionallysubstituted aryl; R³ and R⁴ are each independently a hydrogen, anoptionally substituted alkyl, an optionally substituted cycloalkyl, anoptionally substituted arylalkyl, an optionally substituted aryl, anoptionally substituted heterocyclyl, an optionally substituted alkoxy, ahydroxyl, a halo, a nitro, a cyano, a N-monosubstituted amino group, ora N,N-disubstituted amino group; and X is Cl, Br, or I.
 2. The gold(III)complex of formula (I) of claim 1, wherein R¹ and R² are eachindependently a C₁ to C₈ alkyl or a C₇ to C₁₂ arylalkyl.
 3. Thegold(III) complex of formula (I) of claim 1, wherein R¹ and R² are eachmethyl, ethyl, or benzyl.
 4. The gold(III) complex of formula (I) ofclaim 1, wherein R³ and R⁴ are each hydrogen.
 5. The gold(III) complexof formula (I) of claim 1, wherein X is Cl.
 6. The gold(III) complex offormula (I) of claim 1, which is selected from the group consisting of


7. A pharmaceutical composition, comprising: the gold(III) complex offormula (I) of claim 1; and a pharmaceutically acceptable carrier and/orexcipient.
 8. The pharmaceutical composition of claim 7, wherein thegold(III) complex of formula (I) is present in the pharmaceuticalcomposition in a concentration of 1 to 50 μM, relative to a total volumeof the pharmaceutical composition.
 9. A method for treating cancer in asubject, comprising: administering to the subject a therapeuticallyeffective amount of the gold(III) complex of formula (I) of claim 1,wherein the cancer is at least one selected from the group consisting ofbone cancer, lung cancer, prostate cancer, breast cancer, ovariancancer, and cervical cancer.
 10. The method of claim 9, wherein thetherapeutically effective amount of the gold(III) complex of formula (I)is from 0.01 to 25 mg/kg of the gold(III) complex of formula (I) perbody weight of the subject.
 11. A gold(III) complex of formula (II),

or a pharmaceutically acceptable salt, solvate, tautomer, orstereoisomer thereof, wherein: R¹ and R² are each independently ahydrogen, an optionally substituted alkyl, an optionally substitutedcycloalkyl, an optionally substituted arylalkyl, or an optionallysubstituted aryl; R³ and R⁴ are each independently an optionallysubstituted alkyl, an optionally substituted cycloalkyl, an optionallysubstituted arylalkyl, an optionally substituted aryl, an optionallysubstituted heterocyclyl, an optionally substituted alkoxy, a hydroxyl,a halo, a nitro, a cyano, a N-monosubstituted amino group, or aN,N-disubstituted amino group; and X is Cl, Br, or I.
 12. The gold(III)complex of formula (II) of claim 11, wherein R¹ and R² are eachindependently a C₁ to C₈ alkyl or a C₇ to C₁₂ arylalkyl.
 13. Thegold(III) complex of formula (II) of claim 11, wherein R¹ and R² areeach methyl, ethyl, or benzyl.
 14. The gold(III) complex of formula (II)of claim 11, wherein R³ and R⁴ are each hydroxyl.
 15. The gold(III)complex of formula (II) of claim 11, wherein X is Cl.
 16. The gold(III)complex of formula (II) of claim 11, which is selected from the groupconsisting of


17. A pharmaceutical composition, comprising: the gold(III) complex offormula (II) of claim 11; and a pharmaceutically acceptable carrierand/or excipient.
 18. The pharmaceutical composition of claim 17,wherein the gold(III) complex of formula (II) is present in thepharmaceutical composition in a concentration of 1 to 50 μM, relative toa total volume of the pharmaceutical composition.
 19. A method fortreating cancer in a subject, comprising: administering to the subject atherapeutically effective amount of the gold(III) complex of formula(II) of claim 11, wherein the cancer is at least one selected from thegroup consisting of bone cancer, lung cancer, prostate cancer, breastcancer, ovarian cancer, and cervical cancer.
 20. The method of claim 19,wherein the therapeutically effective amount of the gold(III) complex offormula (II) is from 0.01 to 25 mg/kg of the gold(III) complex offormula (II) per body weight of the subject.